WO2017012661A1 - Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents - Google Patents

Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents Download PDF

Info

Publication number
WO2017012661A1
WO2017012661A1 PCT/EP2015/066773 EP2015066773W WO2017012661A1 WO 2017012661 A1 WO2017012661 A1 WO 2017012661A1 EP 2015066773 W EP2015066773 W EP 2015066773W WO 2017012661 A1 WO2017012661 A1 WO 2017012661A1
Authority
WO
WIPO (PCT)
Prior art keywords
scv20265
antibiotic
pseudomonas
antimicrobial
mutation
Prior art date
Application number
PCT/EP2015/066773
Other languages
French (fr)
Inventor
Andreas Keller
Susanne Schmolke
Cord Friedrich Stähler
Christina Backes
Valentina GALATA
Original Assignee
Curetis Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curetis Gmbh filed Critical Curetis Gmbh
Priority to PCT/EP2015/066773 priority Critical patent/WO2017012661A1/en
Priority to PCT/EP2016/067406 priority patent/WO2017013204A1/en
Priority to CN201680038539.3A priority patent/CN108513589A/en
Priority to CA2990908A priority patent/CA2990908A1/en
Priority to AU2016295122A priority patent/AU2016295122A1/en
Priority to US15/745,633 priority patent/US20180265913A1/en
Priority to EP16745655.7A priority patent/EP3325655A1/en
Publication of WO2017012661A1 publication Critical patent/WO2017012661A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicrobial drug treatment, a method of se ⁇ lecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, and a method of determining an antimicrobial drug, e.g. antibiotic, re- sistance profile for bacterial microorganisms of Pseudomonas species, as well as computer program products used in these methods .
  • an antimicrobial drug e.g. antibiotic, re- sistance profile for bacterial microorganisms of Pseudomonas species
  • Antibiotic resistance is a form of drug resistance whereby a sub-population of a microorganism, e.g. a strain of a bacterial species, can survive and multiply despite exposure to an antibiotic drug. It is a serious and health concern for the individual patient as well as a major public health issue. Timely treatment of a bacterial infection requires the analy ⁇ sis of clinical isolates obtained from patients with regard to antibiotic resistance, in order to select an efficacious therapy. Generally, for this purpose an association of the identified resistance with a certain microorganism (i.e. ID) is necessary.
  • Antibacterial drug resistance represents a major health burden. According to the World Health Organization's antimicrobial resistance global report on surveillance, ADR leads to 25,000 deaths per year in Europe and 23,000 deaths per year in the US. In Europe, 2.5 million extra hospital days lead to societal cost of 1.5 billion euro. In the US, the di ⁇ rect cost of 2 million illnesses leads to 20 billion dollar direct cost. The overall cost is estimated to be substantial- ly higher, reducing the gross domestic product (GDP) by up to Pseudomonas ssp. are gram-negative, aerobic bacilli belonging to the family of Pseudomonadaceae . Pseudomonas aeruginosa has received the most attention because of the frequency with which it is involved in human disease.
  • GDP gross domestic product
  • Pseudomonas aeruginosa causes various diseases.
  • the pathogen is increasingly recognized as an important etiology of healthcare-associated pneumonia and is consistently identi ⁇ fied as the most commonly isolated pathogen causing ventila ⁇ tor-associated pneumonia.
  • Pseudomonas aeruginosa is well known as a cause of chronic infection of the lungs and airways in patients with cystic fibrosis. Localized in- fection following surgery or burns commonly results in a generalized and frequently fatal bacteremia.
  • Urinary tract in ⁇ fections following introduction of Pseudomonas aeruginosa on catheters or in irrigating solutions are not uncommon.
  • Pseu ⁇ domonas aeruginosa can cause severe corneal infections fol- lowing eye surgery or injury. It occasionally causes meningitis following lumbar puncture and endocarditis following cardiac surgery. It has been associated with some diarrheal dis ⁇ ease episodes.
  • Pseudomonas is intrinsically resistant to a multitude of an ⁇ tibiotics presumably as a result of impermeability of the outer membrane combined with active efflux pumps. Besides in ⁇ trinsic resistance, Pseudomonas easily develops acquired re ⁇ sistance either by mutation in chromosomally encoded genes or by the horizontal gene transfer of antibiotic resistance de ⁇ terminants . In a recent report by CDC, titled Antibiotic Resistance
  • Efflux pumps are high-affinity reverse transport systems located in the membrane that transports the antibiotic out of the cell, e.g. resistance to tetracycline.
  • the penicillinases are a group of beta-lactamase enzymes that cleave the beta lactam ring of the penicillin molecule.
  • pathogens show natural resistance against drugs.
  • an organism can lack a transport system for an antibiotic or the target of the antibiotic molecule is not present in the organism.
  • Pathogens that are in principle susceptible to drugs can be- come resistant by modification of existing genetic material (e.g. spontaneous mutations for antibiotic resistance, hap ⁇ pening in a frequency of one in about 100 mio bacteria in an infection) or the acquisition of new genetic material from another source.
  • existing genetic material e.g. spontaneous mutations for antibiotic resistance, hap ⁇ pening in a frequency of one in about 100 mio bacteria in an infection
  • Horizontal gene transfer may happen by transduction, transformation or conj ugation .
  • testing for susceptibility/resistance to antimi- crobial agents is performed by culturing organisms in differ ⁇ ent concentration of these agents.
  • agar plates are inoculated with patient sample (e.g. urine, sputum, blood, stool) overnight.
  • patient sample e.g. urine, sputum, blood, stool
  • individual colonies are used for identification of organisms, either by culturing or using mass spectroscopy.
  • new plates containing increasing concentration of drugs used for the treatment of these organisms are inoculated and grown for additional 12 - 24 hours.
  • the lowest drug concentration which inhibits growth is used to determine suscepti ⁇ bility/resistance for tested drugs.
  • the process takes at least 2 to 3 working days during which the patient is treated empirically. A significant reduction of time-to-result is needed especially in patients with life-threatening disease and to overcome the widespread misuse of antibiotics.
  • targets include DNA Topoisomerase IV, DNA Topoisomerase II and DNA Gyrase. It can be expected that this is also the case for other drugs alt ⁇ hough the respective secondary targets have not been identi ⁇ fied yet. In case of a common regulation, both relevant ge ⁇ netic sites would naturally show a co-correlation or redundancy .
  • Wozniak et al (BMC Genomics 2012, 13 (Suppl 7):S23) disclose genetic determinants of drug resistance in Staphylococcus aureus based on genotype and phenotype data.
  • Stoesser et al disclose prediction of antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data (J Antimicrob Chemother 2013; 68: 2234-2244) .
  • Chewapreecha et al (Chewapreecha et al (2014) Comprehensive Identification of single nucleotid polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes.
  • PLoS Genet 10(8) : el004547) used a comparable approach to identify mutations in gram-positive Streptococcus Pneumonia.
  • the present inventors addressed this need by carrying out whole genome sequencing of a large cohort of Pseudomonas clinical isolates and comparing the genetic mutation profile to classical culture based antimicrobial susceptibility test ⁇ ing with the goal to develop a test which can be used to de ⁇ tect bacterial susceptibility/resistance against antimicrobi ⁇ al drugs using molecular testing.
  • the inventors performed extensive studies on the genome of bacteria of Pseudomonas species either susceptible or re ⁇ sistant to antimicrobial, e.g. antibiotic, drugs. Based on this information, it is now possible to provide a detailed analysis on the resistance pattern of Pseudomonas strains based on individual genes or mutations on a nucleotide level. This analysis involves the identification of a resistance against individual antimicrobial, e.g. antibiotic, drugs as well as clusters of them. This allows not only for the deter- mination of a resistance to a single antimicrobial, e.g. an ⁇ tibiotic, drug, but also to groups of antimicrobial drugs, e.g.
  • the present invention will considerably facilitate the selection of an appropriate antimicrobial, e.g. antibi ⁇ otic, drug for the treatment of a Pseudomonas infection in a patient and thus will largely improve the quality of diagno ⁇ sis and treatment.
  • an appropriate antimicrobial e.g. antibi ⁇ otic
  • the present invention discloses a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicro ⁇ bial drug treatment, which can be also described as a method of determining an antimicrobial drug, e.g. antibiotic, re ⁇ sistant Pseudomonas infection of a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa ⁇ tient ;
  • An infection of a patient with Pseudomonas species potential ⁇ ly resistant to antimicrobial drug treatment herein means an infection of a patient with Pseudomonas species wherein it is unclear if the Pseudomonas species is susceptible to treat- ment with a specific antimicrobial drug or if it is resistant to the antimicrobial drug.
  • step b) above as well as corresponding steps, at least one mutation in at least two genes is determined, so that in total at least two mutations are determined, wherein the two mutations are in different genes.
  • the present invention relates to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, e.g. from an antimicrobial drug, e.g. antibiotic, re ⁇ sistant Pseudomonas infection, comprising the steps of:
  • the present invention disclos ⁇ es a computer program product comprising executable instruc ⁇ tions which, when executed, perform a method according to the third, fourth, fifth, sixth or seventh aspect of the present invention .
  • Further aspects and embodiments of the invention are dis ⁇ closed in the dependent claims and can be taken from the fol ⁇ lowing description, figures and examples, without being limited thereto.
  • Fig. 1 shows schematically a read-out concept for a diagnos- tic test according to a method of the present invention.
  • mutation relates to a variation in the sequence as compared to a reference sequence.
  • a reference sequence can be a sequence determined in a predominant wild type or- ganism or a reference organism, e.g. a defined and known bac ⁇ terial strain or substrain.
  • a mutation is for example a deletion of one or multiple nucleotides, an insertion of one or multiple nucleotides, or substitution of one or multiple nu ⁇ cleotides, duplication of one or a sequence of multiple nu- cleotides, translocation of one or a sequence of multiple nu ⁇ cleotides, and, in particular, a single nucleotide polymor ⁇ phism (SNP) .
  • SNP single nucleotide polymor ⁇ phism
  • sample is a sam- pie which comprises at least one nucleic acid molecule from a bacterial microorganism.
  • samples are: cells, tissue, body fluids, biopsy specimens, blood, urine, saliva, sputum, plasma, serum, cell culture supernatant, swab sample and others.
  • the sample is a patient sample (clinical isolate) .
  • mutations in at least two, three, four, five, six, seven, eight, nine or ten genes are determined in any of the methods of the present invention, e.g. in at least two genes or in at least three genes.
  • a combination of several variant positions can improve the prediction accu- racy and further reduce false positive findings that are in ⁇ fluenced by other factors. Therefore, it is in particular preferred to determine the presence of a mutation in 2, 3, 4, 5, 6, 7, 8 or 9 (or more) genes selected from Table 1 or 2.
  • Tables 1 and 2 the highest probability of a resistance to at least one antimicrobial drug, e.g.
  • nucleic acids and/or nucle ⁇ ic acid fragments and/or parts thereof contained therein in a short period of time including the nucleic acids and/or nu ⁇ cleic acid fragments and/or parts thereof of at least one mi- croorganism of interest, particularly of at least one Pseudo ⁇ monas species.
  • sequencing can be carried out us ⁇ ing polymerase chain reaction (PCR) , particularly multiplex PCR, or high throughput sequencing or next generation sequencing, preferably using high-throughput sequencing.
  • PCR polymerase chain reaction
  • sequencing preferably an in vitro sample is used.
  • dif- ferent reference genomes or more than one reference genomes can be used for aligning.
  • the reference genome - as well as also the data from the genomes of the other species, e.g. Pseudomonas species - mutations in the genes for each species and for the whole multitude of samples of different species, e.g. Pseudomonas species, can be obtained.
  • RefSeq RefSeq
  • matrices % of mapped reads, % of covered genome
  • the reference sequence was obtained from Pseudomonas strain NC_023149 (http : //www . genome . jp/dbget- bin/www_bget?refseq+NC_023149)
  • the gene sequence of the first data set can be assembled, at least in part, with known meth ⁇ ods, e.g. by de-novo assembly or mapping assembly.
  • the se ⁇ quence assembly is not particularly limited, and any known genome assembler can be used, e.g. based on Sanger, 454, Solexa, Illumina, SOLid technologies, etc., as well as hy ⁇ brids/mixtures thereof.
  • the data of nucleic acids of different origin than the microorganism of interest can be removed after the nucleic acids of interest are identified, e.g. by filtering the data out.
  • Such data can e.g. include nucleic acids of the patient, e.g. the vertebrate, e.g. human, and/or other microorganisms, etc. This can be done by e.g. computational subtraction, as devel- oped by Meyerson et al . 2002. For this, also aligning to the genome of the vertebrate, etc., is possible. For aligning, several alignment-tools are available. This way the original data amount from the sample can be drastically reduced.
  • fingerprinting and/or aligning, and/or assembly, etc. can be carried out, as described above, forming a third data set of aligned and/or assembled genes for a Pseudomonas species.
  • genes with mutations of the microor ⁇ ganism of interest e.g. Pseudomonas species
  • genes with mutations of the microor ⁇ ganism of interest e.g. Pseudomonas species
  • susceptibility of a number of antimicrobial drugs e.g. antibiotics, e.g. using standard culturing meth ⁇ ods on dishes with antimicrobial drug, e.g. antibiotic, in- take, as e.g.
  • the results of these antimi ⁇ crobial drug, e.g. antibiotic, susceptibility tests can then be cross-referenced/correlated with the mutations in the ge ⁇ nome of the respective microorganism, e.g. Pseudomonas .
  • the mutations in the ge ⁇ nome of the respective microorganism e.g. Pseudomonas .
  • antimicrobial drug
  • samples can be e.g. cultured overnight. On the next day individual colonies can be used for identification of organisms, either by culturing or using mass spectroscopy. Based on the identity of organisms new plates containing increasing concentration of antibiotics used for the treatment of these organisms are inoculated and grown for additional 12 - 24 hours. The lowest drug concen- tration which inhibits growth (minimal inhibitory concentra ⁇ tion - MIC) can be used to determine susceptibil ⁇ ity/resistance for tested antibiotics.
  • Correlation of the nucleic acid / gene mutations with antimi- crobial drug, e.g. antibiotic, resistance can be carried out in a usual way and is not particularly limited.
  • resistances can be correlated to certain genes or certain mu ⁇ tations, e.g. SNPs, in genes.
  • statistical analysis can be carried out.
  • statistical analysis of the correlation of the gene mutations with antimicrobial drug, e.g. antibiotic, re ⁇ sistance is not particularly limited and can be carried out, depending on e.g.
  • the amount of data in different ways, for example using analysis of variance (ANOVA) or Student's t- test, for example with a sample size n of 50, 100, 200, 300, 400, 500, e.g. 1000 or 1100, and a level of significance ( - error-level) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller.
  • a statistical value can be obtained for each gene and/or each position in the genome as well as for all antibiotics tested, a group of antibiotics or a single anti ⁇ biotic.
  • the obtained p-values can also be adapted for statis ⁇ tical errors, if needed.
  • n 50, 100, 200, 300, 400 or 500, e.g. 1000 or 1100, and a level of significance ( -error- level) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller.
  • a level of significance e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller.
  • n 50 or more, 100 or more, 200 or more, 300 or more, 400 or more or 500 or more, e.g. 1000 or more or 1100 or more, and a level of significance ( -error- level) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller.
  • a level of significance e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller.
  • the present invention relates in a second aspect to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, e.g. from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
  • SCV20265 _5597, and SCV20265 0241 wherein the presence of said at least two mutations is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;
  • step c) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection.
  • antimicrobial e.g. antibiotic
  • the steps a) of obtaining or providing a sam- pie and b) of determining the presence of at least one muta ⁇ tion are as in the method of the first aspect.
  • the identification of the at least one or more antimicrobial, e.g. antibiotic, drug in step c) is then based on the results obtained in step b) and corresponds to the antimicrobial, e.g. antibiotic, drug(s) that correlate (s) with the muta ⁇ tions.
  • the antimicrobial drugs e.g. antibiotics
  • the remaining antimicrobial drugs can be selected in step d) as being suita- ble for treatment.
  • references to the first and second aspect also apply to the 14 th , 15 th , 16 th and 17 th aspect, referring to the same genes, unless clear from the context that they don't apply.
  • NC_023149 as annotated at the NCBI is determined.
  • a particularly relevant correlation with antimicrobial drug, e.g. antibiotic, resistance could be deter- mined.
  • the mutation in position 1979239 with regard to reference genome NC_023149 as annotated at the NCBI is a non-synonymous coding, particularly a codon change aCc/aTc; aCc/aAc, and the mutation in position 5987559 with re- gard to reference genome NC_023149 as annotated at the NCBI is a non-synonymous coding, particularly a codon change tCg/tTg; tCg/tGg.
  • the antimicrobial drug e.g. antibiotic
  • the antimicrobial drug in the method of the first or second aspect, as well as in the other methods of the invention, is at least one selected from the group of ⁇ -lactams, ⁇ -lactam inhibi ⁇ tors, quinolines and derivatives thereof, aminoglycosides, polyketides, respectively tetracyclines, and folate synthesis inhibitors.
  • the resistance of Pseudomonas to one or more antimicrobial, e.g. antibiotic, drugs can be determined according to certain embodiments.
  • the antimicrobial drug is an antibiotic/antibiotic drug.
  • the antimicrobial, e.g. antibiotic, drug is selected from lactam antibiotics, and the presence of a mutation in the following genes is determined:
  • SCV20265_1892 SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_1879, SCV20265_5242 , SCV20265_2224 , SCV20265_0530 , SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958, SCV20265_3248, SCV20265_1451 , SCV20265_6120 , SCV20265_4839, SCV20265_2195 , SCV20265_0968 , SCV20265_2464, SCV20265 2518, SCV20265 2654, SCV20265 3101, SCV20265 1805, SCV20265_4445, SCV20265_2883 , SCV20265_1721 , SCV20265_3099 , SCV20265_1735, SCV20265_6289 , SCV20265_
  • the antimicrobial, e.g. antibiotic, drug is selected from quinolone antibiotics, e.g.
  • the antimicrobial e.g.
  • SCV20265_1892 SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_3294, SCV20265_1879 , SCV20265_5242 , SCV20265_2224 , SCV20265_0530, SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958 , SCV20265_3248 , SCV20265_1132 , SCV20265 1451, SCV20265 6120, SCV20265 4839, SCV20265 2195, SCV20265_0968, SCV20265_2464, SCV20265_2518, SCV20265_2654, SCV20265_3101, SCV20265_3909, SCV20265_2610, SCV20265_1805, SCV20265_1892692, SCV20265_5625 , SCV20265_1467 , SC
  • the antimicrobial, e.g. antibiotic, drug is selected from other antibiotics ( (benzene de ⁇ rived) /sulfonamide) , and the presence of a mutation in the following genes is determined: SCV20265_1892 , SCV20265_5625,
  • determining the nucleic acid se ⁇ quence information or the presence of a mutation comprises determining the presence of a single nucleotide at a single position in a gene.
  • the invention comprises methods wherein the presence of a single nucleotide polymorphism or mutation at a single nucleotide position is detected.
  • the antibiotic drug in the methods of the present invention is selected from the group consisting of Amoxicillin/K Clavulanate (AUG) , Ampicillin (AM), Aztreonam (AZT) , Cefazolin (CFZ) , Cefepime (CPE),
  • SCV20265 _1892 SCV20265 _5625, SCV20265 _1467, SCV20265 _5607,
  • the gene is from Table 1 or Table 2
  • the antibiotic drug is selected from lactam antibiotics and a mutation in at least one of the following nucleotide posi ⁇ tions is detected with regard to reference genome NC_023149: 1979239, 5987559, 1537406, 5965080, 1967346, 5569783 2350860, 562872, 3507580, 1947689, 2316386, 6685845, 3142437 3468647, 1521674, 6520799, 5124971, 2317909, 1009933 2567532, 2611669, 2754829, 3301233, 1899865, 4712288 3019764, 1805165, 3299685, 1821163, 6702956, 3160788 6535290, 3881624, 1099519, 5662982, 2903129, 2363393 2350862, 3507601, 3507667, 6519971
  • the gene is from Table 1 or Table 2
  • the antibiotic drug is selected from quinolone antibiotics, e.g. fluoroquinolone antibiotics, and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149: 1979239, 5987559,
  • the antibiotic drug is P/T and a mu tation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149:
  • the antibiotic drug is CFT, IMP, MER, CAX, AZT, and/or CAZ and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149: 1979239, 5987559.
  • the resistance of a bacterial micro ⁇ organism belonging to the species Pseudomonas against 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, 17, 18, 19, 20 or 21 antibiotic drugs is determined.
  • determining the nucleic acid se- quence information or the presence of a mutation comprises determining a partial or entire sequence of the genome of the Pseudomonas species, wherein said partial or entire sequence of the genome comprises at least a partial sequence of said at least two genes.
  • determining the nucleic acid se ⁇ quence information or the presence of a mutation comprises using a next generation sequencing or high throughput sequencing method.
  • a partial or en ⁇ tire genome sequence of the bacterial organism of Pseudomonas species is determined by using a next generation sequencing or high throughput sequencing method.
  • the second da- ta set e.g. comprises, respectively is, a set of antimicrobi ⁇ al drug, e.g. antibiotic, resistances of a plurality of clin ⁇ ical isolates
  • this can, within the scope of the invention, also refer to a self-learning data base that, whenever a new sample is analyzed, can take this sample into the second data set and thus expand its data base.
  • the second data set thus does not have to be static and can be expanded, either by ex ⁇ ternal input or by incorporating new data due to self- learning.
  • the method of the third aspect of the present invention can, according to certain embodiments, comprise cor ⁇ relating different genetic sites to each other. This way even higher statistical significance can be achieved.
  • the second data set is provided by culturing the clinical isolates of Pseudomonas species on agar plates provided with antimicrobial drugs, e.g. antibiotics, at different concentrations and the second data is obtained by taking the minimal concentration of the plates that inhibits growth of the respective Pseudomonas species .
  • the antibiotic is at least one selected from the group of ⁇ -lactams, ⁇ -lactam inhibitors, quinolines and derivatives thereof, aminoglycosides,
  • tetracyclines and folate synthesis inhibitors, preferably
  • Amoxicillin/K Clavulanate Ampicillin, Aztreonam, Cefazolin, Cefepime, Cefotaxime, Ceftazidime, Ceftriaxone, Cefuroxime, Cephalothin, Ciprofloxacin, Ertapenem, Gentamicin, Imipenem, Levofloxacin, Meropenem, Piperacillin/Tazobactam, Ampicil- lin/Sulbactam, Tetracycline, Tobramycin, and Trimethoprim/Sulfamethoxazole .
  • the gene sequences in the third data set are comprised in at least one gene from the group of genes consisting of SCV20265_1892 , SCV20265_5625,
  • the genetic variant has a point mutation, an insertion and or deletion of up to four bases, and/or a frameshift mutation, particularly a non-synonymous coding in YP 008980900.1 and/or YP 008984625.1.
  • a fourth aspect of the present invention relates to a method of determining an antimicrobial drug, e.g. antibiotic, re ⁇ sistance profile for a bacterial microorganism belonging to the species Pseudomonas comprising the steps of
  • Steps a) and b) can herein be carried out as described with regard to the first aspect, as well as for the following as ⁇ pects of the invention.
  • any mutations in the genome of Pseudomonas species correlated with antimicrobial drug, e.g. antibiotic, resistance can be determined and a thorough antimicrobial drug, e.g. antibiotic, resistance profile can be established
  • antimicrobial drug e.g. antibiotic
  • FIG. 1 A simple read out concept for a diagnostic test as described in this aspect is shown schematically in Fig. 1.
  • a sample 1 e.g. blood from a patient
  • molecular testing 2 e.g. using next generation sequencing (NGS)
  • a molecular fingerprint 3 is taken, e.g. in case of NGS a sequence of selected ge- nomic/plasmid regions or the whole genome is assembled.
  • NGS next generation sequencing
  • a reference library 4 i.e. selected se- quences or the whole sequence are/is compared to one or more reference sequences, and mutations (SNPs, sequence- gene ad ⁇ ditions/deletions, etc.) are correlated with susceptibility/ reference profile of reference strains in the reference li ⁇ brary.
  • the reference library 4 herein contains many genomes and is different from a reference genome. Then the result 5 is reported comprising ID (pathogen identification), i.e. a list of all (pathogenic) species identified in the sample, and AST (antimicrobial susceptibility testing), i.e. a list including a susceptibility /resistance profile for all spe- cies listed
  • ID pathogen identification
  • AST antimicrobial susceptibility testing
  • a fifth aspect of the present invention relates to a diagnos ⁇ tic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicrobial drug treatment, which also can be described as method of de ⁇ termining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection in a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the spe- cies Pseudomonas from the patient;
  • steps a) and b) can herein be carried out as described with regard to the first aspect of the present invention.
  • a Pseudomonas infection in a pa- tient can be determined using sequencing methods as well as a resistance to antimicrobial drugs, e.g. antibiotics, of the Pseudomonas species be determined in a short amount of time compared to the conventional methods.
  • the present invention relates to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, e.g. an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
  • step c) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection.
  • This method can be carried out similarly to the second aspect of the invention and enables a fast was to select a suitable treatment with antibiotics for any infection with an unknown Pseudomonas species.
  • a seventh aspect of the present invention relates to a method of acquiring, respectively determining, an antimicrobial drug, e.g. antibiotic, resistance profile for a bacterial mi ⁇ croorganisms of Pseudomonas species, comprising:
  • antimicrobial drug e.g. antibiotic
  • re ⁇ sistances in an unknown isolate of Pseudomonas can be deter- mined.
  • the reference genome of Pseudomonas is NC_023149 as annotated at the NCBI .
  • statistical analysis in the present methods is carried out using Fisher's test with p ⁇ 10 ⁇ 6 , preferably p ⁇ 10 ⁇ 9 , particularly p ⁇ 10 ⁇ 10 .
  • the method further comprises corre- lating different genetic sites to each other.
  • An eighth aspect of the present invention relates to a com ⁇ puter program product comprising computer executable instructions which, when executed, perform a method according to the third, fourth, fifth, sixth or seventh aspect of the present invention .
  • the computer program product is one on which program commands or program codes of a computer program for executing said method are stored.
  • the computer program product is a storage medium.
  • the computer program prod- ucts of the present invention can be self-learning, e.g. with respect to the first and second data sets.
  • the proposed principle is based on a combination of different approaches, e.g. alignment with at least one, preferably more reference genomes and/or assembly of the genome and correla ⁇ tion of mutations found in every sample, e.g. from each pa ⁇ tient, with all references and drugs, e.g. antibiotics, and search for mutations which occur in several drug and several strains .
  • a list of mutations as well of genes is generated. These can be stored in databases and statistical models can be derived from the databases. The statistical models can be based on at least one or more mutations at least one or more genes. Statistical models that can be trained can be combined from mutations and genes. Examples of algorithms that can produce such models are association
  • the goal of the training is to allow a reproducible, stand- ardized application during routine procedures.
  • a genome or parts of the genome of a microorganism can be sequenced from a patient to be diag ⁇ nosed. Afterwards, core characteristics can be derived from the sequence data which can be used to predict resistance. These are the points in the database used for the final mod ⁇ el, i.e. at least one mutation or at least one gene, but also combinations of mutations, etc. The corresponding characteristics can be used as input for the statistical model and thus enable a prognosis for new pa ⁇ tients. Not only the information regarding all resistances of all microorganisms, e.g. of Pseudomonas species, against all drugs, e.g.
  • a ninth aspect of the present invention relates to the use of the computer program product according to the eighth aspect for acquiring an antimicrobial drug, e.g. antibiotic, re ⁇ sistance profile for bacterial microorganisms of Pseudomonas species or in a method of the third aspect of the invention.
  • an antimicrobial drug e.g. antibiotic, re ⁇ sistance profile for bacterial microorganisms of Pseudomonas species or in a method of the third aspect of the invention.
  • a method of selecting a treatment of a pa ⁇ tient having an infection with a bacterial microorganism of Pseudomonas species comprising:
  • antimicrobial drug e.g. antibiotic, resistance
  • the steps can be carried out as similar steps before.
  • no aligning is nec ⁇ essary, as the unknown sample can be directly correlated, af ⁇ ter the genome or genome sequences are produced, with the se ⁇ cond data set and thus mutations and antimicrobial drug, e.g. antibiotic, resistances can be determined.
  • the first data set can be assembled, for example, using known techniques.
  • statistical analysis in the present method is carried out using Fisher' s test with p ⁇ 10 ⁇ 6 , preferably p ⁇ 10 ⁇ 9 , particularly p ⁇ 10 ⁇ 10 . Also, ac- cording to certain embodiments, the method further comprises correlating different genetic sites to each other.
  • An eleventh aspect of the present invention is directed to a computer program product comprising computer executable in- structions which, when executed, perform a method according to the tenth aspect.
  • a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicrobial drug treatment which can also be described as a method of determining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection of a patient is disclosed, comprising the steps of:
  • an antimicrobial drug e.g. antibiotic, resistant Pseudomonas infection in said patient.
  • a thirteenth aspect of the invention discloses a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infec- tion, comprising the steps of:
  • SCV20265_1892 SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265 3294, SCV20265 1879, SCV20265 5242, SCV20265 2224, SCV20265 _0530, SCV20265 _3289, SCV20265 _1858, SCV20265 _2193,
  • mutations in at least two, three, four, five, six, seven, eight, nine or ten genes are determined in any of the methods of the present invention, e.g. in at least two genes or in at least three genes.
  • a combination of several variant positions can improve the prediction accu ⁇ racy and further reduce false positive findings that are in ⁇ fluenced by other factors. Therefore, it is in particular preferred to determine the presence of a mutation in 2, 3, 4, 5, 6, 7, 8 or 9 (or more) genes selected from Table 5.
  • FDR determined according to FDR (Benjamini Hochberg) method (Benjamini
  • SCV20265_1756, SCV20265_1113 , SCV20265_1895 , SCV20265_4827 is detected, or a mutation in at least one of the positions of
  • SCV20265_0891, SCV20265_1756, SCV20265_1113 , SCV20265_4827 , SCV20265 4562 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 4604211, 938801, 1846678, 1169401, 5100757, 4832599.
  • SCV20265_0891, SCV20265_1756, SCV20265_1113 , SCV20265_1467 , SCV20265_2654, SCV20265_1050 , SCV20265_4562 is detected, or a mutation in at least one of the positions of 1979239,
  • the antibiotic is P/T and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_4334,
  • SCV20265_6289, SCV20265_3626, SCV20265_4159 is detected, or a mutation in at least one of the positions of 1979239,
  • the antibiotic is CPE and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_4334,
  • SCV20265_1895, SCV20265_1467, SCV20265_2654 , SCV20265_6289 , SCV20265_3626, SCV20265_1050, SCV20265_4159 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 4604211, 1984529, 1537406, 2754829, 6702956,
  • the antibiotic is AZT and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_4334 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 4604211.
  • the antibiotic is at least one of CFT, CAX and CAZ and a mutation in at least one of the genes of SCV20265_1892 , SCV20265_5625 is detected, or a mutation in at least one of the positions ofl979239, 5987559.
  • the antibiotic is a quinolone antibiotic and a mutation in at least one of the genes listed in Table 7 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 7.
  • ETP ETP; MER; CAX; AZT; P/T; CPE;
  • ETP ETP; MER; CAX; AZT; P/T; CPE;
  • the antibiotic is at least one of CP and LVX and a mutation in at least one of the genes of SCV20265_1892 , SCV20265_5625,
  • SCV20265_1467, SCV20265_5607 , SCV20265_3294 , SCV20265_1879 , SCV20265_5242, SCV20265_2224 , SCV20265_0530 , SCV20265_3289 , SCV20265_1858, SCV20265_2193 , SCV20265_6274 , SCV20265_2958 , SCV20265_3248 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 1537406, 5965080, 3513162, 1967346, 5569783, 2350860, 2350862, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647.
  • the antibiotic is an aminoglycoside antibiotic and a mutation in at least one of the genes listed in Table 8 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 8.
  • Table 8 List of aminoglycoside antibiotics gene name POS antibiotic p-value genbank protein
  • ETP ETP; MER; CAX; AZT; P/T; CPE;
  • ETP ETP; MER; CAX; AZT; P/T; CPE;
  • the antibiotic is at least one of GM and TO and a mutation in at least one of the genes of SCV20265_1892 , SCV20265_5625,
  • SCV20265_1467, SCV20265_5607 , SCV20265_3294 , SCV20265_1879 , SCV20265_5242, SCV20265_2224 , SCV20265_0530 , SCV20265_3289 , SCV20265_1858, SCV20265_2193 , SCV20265_6274 , SCV20265_2958 , SCV20265_3248 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 1537406, 5965080, 3513162, 1967346, 5569783, 2350860, 2350862, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647.
  • the antibiotic is T/S and a mutation in at least one of the genes listed in Table 9 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 12.
  • a fourteenth aspect of the present invention is directed to a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicro- bial drug treatment, which can also be described as method of determining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection of a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa- tient;
  • SCV20265 _5597, and SCV20265 0241 wherein the presence of said at least one mutation is indicative of an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection in said patient.
  • an antimicrobial drug e.g. antibiotic, resistant Pseudomonas infection in said patient.
  • a fifteenth aspect of the present invention is directed to a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
  • SCV20265_1892 SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_3294, SCV20265_1879 , SCV20265_5242 , SCV20265_2224 , SCV20265_0530, SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958 , SCV20265_3248 , SCV20265_1132 , SCV20265_1451, SCV20265_6120 , SCV20265_4839 , SCV20265_2195 , SCV20265_0968, SCV20265_2464, SCV20265_2518 , SCV20265_2654 , SCV20265_3101, SCV20265_3909 , SCV20265_2610 , SCV20265_1805 , SCV20265 4445, SCV20265 2883, SCV
  • step c) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection.
  • antimicrobial e.g. antibiotic
  • the steps correspond to those in the first or second aspect, although only a mutation in at least one gene is determined.
  • a sixteenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, com ⁇ prising the steps of:
  • an antimicrobial drug e.g. antibiotic, resistant Pseudomonas infection, com ⁇ prising the steps of:
  • SCV20265_1892 SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_3294, SCV20265_1879 , SCV20265_5242 , SCV20265_2224 , SCV20265_0530, SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958 , SCV20265_3248 , SCV20265_1132 , SCV20265_1451, SCV20265_6120 , SCV20265_4839 , SCV20265_2195 , SCV20265_0968, SCV20265_2464, SCV20265_2518 , SCV20265_2654 , SCV20265 3101, SCV20265 3909, SCV20265 2610, SCV20265 1805, SCV20265_4445, SCV20265_2883 , SCV20265_
  • step c) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection; and e) treating the patient with said one or more antimicrobi- al, e.g. antibiotic, drugs.
  • one or more antimicrobial e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection
  • a seventeenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobi ⁇ al drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
  • SCV20265_1892 SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_3294, SCV20265_1879 , SCV20265_5242 , SCV20265_2224 , SCV20265_0530, SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958 , SCV20265_3248 , SCV20265_1132 , SCV20265_1451, SCV20265_6120 , SCV20265_4839 , SCV20265_2195 , SCV20265_0968, SCV20265_2464, SCV20265_2518 , SCV20265_2654 , SCV20265_3101, SCV20265_3909 , SCV20265_2610 , SCV20265_1805 , SCV20265 4445, SCV20265 2883, SCV
  • step c) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection; and e) treating the patient with said one or more antimicrobi ⁇ al, e.g. antibiotic, drugs.
  • one or more antimicrobial e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection
  • An eighteenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobi ⁇ al drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
  • step c) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection; and e) treating the patient with said one or more antimicrobi ⁇ al, e.g. antibiotic, drugs.
  • one or more antimicrobial e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection
  • a nineteenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, com ⁇ prising the steps of:
  • an antimicrobial drug e.g. antibiotic, resistant Pseudomonas infection, com ⁇ prising the steps of:
  • step c) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection; and e) treating the patient with said one or more antimicrobi ⁇ al, e.g. antibiotic, drugs.
  • one or more antimicrobial e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection
  • a twentieth aspect of the present invention is directed to a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicro ⁇ bial drug treatment, which can also be described as method of determining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection of a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa- tient;
  • a twenty-first aspect of the present invention is directed to a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
  • step c) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection.
  • antimicrobial e.g. antibiotic
  • the steps correspond to those in the first or second aspect, although only a mutation in at least one gene is determined. Examples
  • Frozen reference AST panels were prepared following Clinical
  • Isolates were cultured on trypticase soy agar with 5% sheep blood (BBL, Cockeysville, Md.) and incubated in ambient air at 35 ⁇ 1 ° C for 18-24 h. Isolated colonies (4-5 large colonies or 5-10 small colonies) were transferred to a 3 ml Sterile Inoculum Water (Siemens) and emulsified to a final turbidity of a 0.5 McFarland standard. 2 ml of this suspension was add- ed to 25 ml Inoculum Water with Pluronic-F (Siemens) . Using the Inoculator (Siemens) specific for frozen AST panels, 5 ⁇ of the cell suspension was transferred to each well of the AST panel. The inoculated AST panels were incubated in ambi- ent air at 35 ⁇ 1 ° C for 16-20 h. Panel results were read visu ⁇ ally, and minimal inhibitory concentrations (MIC) were deter ⁇ mined .
  • MIC minimal
  • the bacterial isolates Prior to extraction, the bacterial isolates were thawed at room temperature and were pelleted at 2000 G for 5 seconds.
  • the DNA extraction protocol DNAext was used for complete total nucleic acid ex ⁇ traction of 48 isolate samples and eluates, 50 ⁇ each, in 4 hours.
  • the total nucleic acid eluates were then transferred into 96-Well qPCR Detection Plates (401341, Agilent Technolo- gies) for RNase A digestion, DNA quantitation, and plate DNA concentration standardization processes.
  • RNase A (AM2271, Life Technologies) which was diluted in nuclease-free water following manufacturer's instructions was added to 50 ⁇ of the total nucleic acid eluate for a final working concentra- tion of 20 ⁇ g/ml. Digestion enzyme and eluate mixture were incubated at 37 °C for 30 minutes using Siemens VERSANT® Am ⁇ plification and Detection instrument. DNA from the RNase digested eluate was quantitated using the Quant-iTTM PicoGreen dsDNA Assay (P11496, Life Technologies) following the assay kit instruction, and fluorescence was determined on the Sie ⁇ mens VERSANT® Amplification and Detection instrument. Data analysis was performed using Microsoft® Excel 2007.
  • Raw paired-end sequencing data for the 1104 Pseudomonas sam ⁇ ples were mapped against the Pseudomonas reference (NC_023149) with BWA 0.6.1.20.
  • the resulting SAM files were sorted, converted to BAM files, and PCR duplicates were marked using the Picard tools package 1.104
  • the Genome Analysis Toolkit 3.1.1 (GATK)21 was used to call SNPs and indels for blocks of 200 Pseudomonas samples (parameters: -ploidy 1 -glm BOTH - stand_call_conf 30 -stand_emit_conf 10) .
  • VCF files were combined into a single file and quality filtering for SNPs was carried out (QD ⁇ 2.0
  • genotypes of all Pseudomonas samples were consid ⁇ ered.
  • Pseudomonas samples were split into two groups, low re ⁇ sistance group (having lower MIC concentration for the con- sidered drug) , and high resistance group (having higher MIC concentrations) with respect to a certain MIC concentration (breakpoint) .
  • breakpoint a certain MIC concentration
  • the best computed breakpoint was the threshold yielding the lowest p-value for a certain genomic position and drug.
  • positions with non-synonymous alterations and p-value ⁇ 10 were considered.
  • Pseudomonas strains to be tested were seeded on agar plates and incubated under growth conditions for 24 hours. Then, colonies were picked and incubated in growth medium in the presence of a given antibiotic drug in dilution series under growth conditions for 16-20 hours. Bacterial growth was de ⁇ termined by observing turbidity.
  • samples were prepared using a Nextera library preparation, followed by multiplexed sequencing using the Illuminat HiSeq 2500 system, paired end sequencing. Data were mapped with BWA (Li H. and Durbin R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinfor- matics, Epub . [PMID: 20080505] ) and SNP were called using samtools (Li H.*, Handsaker B.*, Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R.
  • the mutations were matched to the genes and the amino acid changes were calculated. Using different algorithms (SVM, ho ⁇ mology modeling) mutations leading to amino acid changes with likely pathogenicity / resistance were calculated.
  • the genetic data were mapped to dif ⁇ ferent reference genomes of Pseudomonas that have been anno ⁇ tated at the NCBI (https://www.ncbi.nlm.nih.gov/), and the best reference was chosen as template for the alignment - NC_023149 as annotated at the NCBI. Additionally, assemblies were carried out and it was verified that the sequenced ge ⁇ nomes fulfil all quality criteria to become reference ge ⁇ nomes . Next, genetic variants were evaluated. This approach resulted in a table with the genetic sites in columns and the same isolates in 1104 rows. Each table entry contained the genetic determinant at the respective site (A, C, T, G, small inser ⁇ tions and deletions, ...) for the respective isolate.
  • Tables 3 and 4a, 4b and 4c A full list of all genetic sites, drugs, drug classes, af ⁇ fected genes etc. is provided in Tables 3 and 4a, 4b and 4c, wherein Table 3 corresponds to Table 1 and represents the genes having the lowest p-values after determining mutations in the genes, and Table 4, respectively Tables 4a, 4b and 4c correspond to Table 2 and represent the genes having the low ⁇ est p-values after correlating the mutations with antibiotic resistance .
  • Tables 5 - 9 the data with the best p-values for each antibi ⁇ otic class with the most antibiotic drugs, respectively, were evaluated, being disclosed in Tables 5 - 9.
  • POS genomic position of the SNP / variant in the Pseudomonas reference genome (see above) ; p-value: significance value calculated using Fishers exact test (determined according to FDR (Benjamini Hochberg) method (Benjamini Hochberg, 1995));
  • NCBI genbank protein accession number of the corresponding protein of the genes
  • the p-value was calculated using the Fisher exact test based on contingency table with 4 fields: #samples Resistant / wild type; #samples Resistant / mutant; #samples not Resistant / wild type; #samples not Resistant / mutant
  • the test is based on the distribution of the samples in the 4 fields. Even distribution indicates no significance, while clustering into two fields indicates significance. The following results were obtained
  • YP_008984625.1 respectively, particular in positions 1979239 and/or 5987559, respectively, with regard to reference genome NC_023149 as annotated at the NCBI; the mutation in position
  • Amoxicillin/Clavulanate Ampicillin, Ampicillin/Sulbactam, Aztreonam, Cefazolin, Cefepime, Ceftazidime, Cefuroxime, Cephalothin, Imipenem, Piperacillin/Tazobactam, Ciprofloxacin, Levofloxacin, Gentamycin, Tobramycin, Tetracycline, Tri- methoprim/Sulfamethoxazol
  • a genetic test for the combined pathogen identification and antimicrobial susceptibility testing direct from the patient sample can reduce the time-to actionable result significantly from several days to hours, thereby enabling targeted treat ⁇ ment. Furthermore, this approach will not be restricted to central labs, but point of care devices can be developed that allow for respective tests. Such technology along with the present methods and computer program products could revolu ⁇ tionize the care, e.g. in intense care units or for admis ⁇ sions to hospitals in general. Furthermore, even applications like real time outbreak monitoring can be achieved using the present methods.
  • the present ap ⁇ proach Compared to approaches using MALDI-TOF MS, the present ap ⁇ proach has the advantage that it covers almost the complete genome and thus enables us to identify the potential genomic sites that might be related to resistance. While MALDI-TOF MS can also be used to identify point mutations in bacterial proteins, this technology only detects a subset of proteins and of these not all are equally well covered. In addition, the identification and differentiation of certain related strains is not always feasible.
  • the present method allows computing a best breakpoint for the separation of isolates into resistant and susceptible groups.
  • the inventors designed a flexible software tool that allows to consider - besides the best breakpoints - also values de ⁇ fined by different guidelines (e.g. European and US guide ⁇ lines) , preparing for an application of the GAST in different countries.
  • the inventors demonstrate that the present approach is capa ⁇ ble of identifying mutations in genes that are already known as drug targets, as well as detecting potential new target sites.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicrobial drug treatment, a method of selecting a treatment of a patient suffering from an antibiotic resistant Pseudomonas infection, and a method of determining an antibiotic resistance profile for bacterial microorganisms of Pseudomonas species, as well as computer program products used in these methods. In an exemplary method, a sample 1, is used for molecular testing 2, and then a molecular fingerprint 3 is taken. The result is then compared to a reference library 4, and the result 5 is reported.

Description

Genetic testing for predicting resistance of Pseudomonas species against antimicrobial agents
The present invention relates to a method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicrobial drug treatment, a method of se¬ lecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, and a method of determining an antimicrobial drug, e.g. antibiotic, re- sistance profile for bacterial microorganisms of Pseudomonas species, as well as computer program products used in these methods .
Antibiotic resistance is a form of drug resistance whereby a sub-population of a microorganism, e.g. a strain of a bacterial species, can survive and multiply despite exposure to an antibiotic drug. It is a serious and health concern for the individual patient as well as a major public health issue. Timely treatment of a bacterial infection requires the analy¬ sis of clinical isolates obtained from patients with regard to antibiotic resistance, in order to select an efficacious therapy. Generally, for this purpose an association of the identified resistance with a certain microorganism (i.e. ID) is necessary.
Antibacterial drug resistance (ADR) represents a major health burden. According to the World Health Organization's antimicrobial resistance global report on surveillance, ADR leads to 25,000 deaths per year in Europe and 23,000 deaths per year in the US. In Europe, 2.5 million extra hospital days lead to societal cost of 1.5 billion euro. In the US, the di¬ rect cost of 2 million illnesses leads to 20 billion dollar direct cost. The overall cost is estimated to be substantial- ly higher, reducing the gross domestic product (GDP) by up to Pseudomonas ssp. are gram-negative, aerobic bacilli belonging to the family of Pseudomonadaceae . Pseudomonas aeruginosa has received the most attention because of the frequency with which it is involved in human disease. Although it seldom causes disease in healthy individuals, it is a major threat to hospitalized patients, particularly those with serious un¬ derlying diseases such as cancer and burns. The high mortali¬ ty associated with these infections is due to a combination of weakened host defenses, bacterial resistance to antibiot- ics, and the production of extracellular bacterial enzymes and toxins .
Pseudomonas aeruginosa causes various diseases. The pathogen is increasingly recognized as an important etiology of healthcare-associated pneumonia and is consistently identi¬ fied as the most commonly isolated pathogen causing ventila¬ tor-associated pneumonia. Furthermore, Pseudomonas aeruginosa is well known as a cause of chronic infection of the lungs and airways in patients with cystic fibrosis. Localized in- fection following surgery or burns commonly results in a generalized and frequently fatal bacteremia. Urinary tract in¬ fections following introduction of Pseudomonas aeruginosa on catheters or in irrigating solutions are not uncommon. Pseu¬ domonas aeruginosa can cause severe corneal infections fol- lowing eye surgery or injury. It occasionally causes meningitis following lumbar puncture and endocarditis following cardiac surgery. It has been associated with some diarrheal dis¬ ease episodes. Pseudomonas is intrinsically resistant to a multitude of an¬ tibiotics presumably as a result of impermeability of the outer membrane combined with active efflux pumps. Besides in¬ trinsic resistance, Pseudomonas easily develops acquired re¬ sistance either by mutation in chromosomally encoded genes or by the horizontal gene transfer of antibiotic resistance de¬ terminants . In a recent report by CDC, titled Antibiotic Resistance
Threats in the United States, 2013, multidrug-resistant Pseu- domonas aeruginosa was listed among bacteria that pose a se¬ rious threat level. Approximately 8% of all healthcare- associated infections reported to CDC s National Healthcare Safety Network are caused by Pseudomonas aeruginosa. About 13% of severe healthcare-associated infections caused by Pseudomonas aeruginosa are multidrug resistant, meaning sev¬ eral classes of antibiotics no longer cure these infections.
In general the mechanisms for resistance of bacteria against antimicrobial treatments rely to a very substantial part on the organism's genetics. The respective genes or molecular mechanisms are either encoded in the genome of the bacteria or on plasmids that can be interchanged between different bacteria. The most common resistance mechanisms include:
1) Efflux pumps are high-affinity reverse transport systems located in the membrane that transports the antibiotic out of the cell, e.g. resistance to tetracycline.
2) Specific enzymes modify the antibiotic in a way that it loses its activity. In the case of streptomycin, the an¬ tibiotic is chemically modified so that it will no long¬ er bind to the ribosome to block protein synthesis.
3) An enzyme is produced that degrades the antibiotic,
thereby inactivating it. For example, the penicillinases are a group of beta-lactamase enzymes that cleave the beta lactam ring of the penicillin molecule.
In addition, some pathogens show natural resistance against drugs. For example, an organism can lack a transport system for an antibiotic or the target of the antibiotic molecule is not present in the organism.
Pathogens that are in principle susceptible to drugs can be- come resistant by modification of existing genetic material (e.g. spontaneous mutations for antibiotic resistance, hap¬ pening in a frequency of one in about 100 mio bacteria in an infection) or the acquisition of new genetic material from another source. One example is horizontal gene transfer, a process where genetic material contained in small packets of DNA can be transferred between individual bacteria of the same species or even between different species. Horizontal gene transfer may happen by transduction, transformation or conj ugation .
Generally, testing for susceptibility/resistance to antimi- crobial agents is performed by culturing organisms in differ¬ ent concentration of these agents.
In brief, agar plates are inoculated with patient sample (e.g. urine, sputum, blood, stool) overnight. On the next day individual colonies are used for identification of organisms, either by culturing or using mass spectroscopy. Based on the identity of organisms new plates containing increasing concentration of drugs used for the treatment of these organisms are inoculated and grown for additional 12 - 24 hours. The lowest drug concentration which inhibits growth (minimal in¬ hibitory concentration - MIC) is used to determine suscepti¬ bility/resistance for tested drugs. The process takes at least 2 to 3 working days during which the patient is treated empirically. A significant reduction of time-to-result is needed especially in patients with life-threatening disease and to overcome the widespread misuse of antibiotics.
Recent developments include PCR based test kits for fast bac¬ terial identification (e.g. Biomerieux Biofire Tests, Curetis Unyvero Tests) . With these test the detection of selected re¬ sistance loci is possible for a very limited number of drugs, but no correlation to culture based AST is given. Mass spec¬ troscopy is increasingly used for identification of pathogens in clinical samples (e.g. Bruker Biotyper) , and research is ongoing to establish methods for the detection of susceptibility/resistance against antibiotics. For some drugs such it is known that at least two targets are addressed, e.g. in case of Ciprofloxacin (drug bank ID 00537; https://www.drugbank.ca/drugs/DB00537) targets include DNA Topoisomerase IV, DNA Topoisomerase II and DNA Gyrase. It can be expected that this is also the case for other drugs alt¬ hough the respective secondary targets have not been identi¬ fied yet. In case of a common regulation, both relevant ge¬ netic sites would naturally show a co-correlation or redundancy .
It is known that drug resistance can be associated with ge¬ netic polymorphisms. This holds for viruses, where resistance testing is established clinical practice (e.g. HIV genotyp- ing) . More recently, it has been shown that resistance has also genetic causes in bacteria and even higher organisms, such as humans where tumors resistance against certain cyto¬ static agents can be linked to genomic mutations.
Wozniak et al . (BMC Genomics 2012, 13 (Suppl 7):S23) disclose genetic determinants of drug resistance in Staphylococcus aureus based on genotype and phenotype data. Stoesser et al . disclose prediction of antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data (J Antimicrob Chemother 2013; 68: 2234-2244) .
Chewapreecha et al (Chewapreecha et al (2014) Comprehensive Identification of single nucleotid polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes. PLoS Genet 10(8) : el004547) used a comparable approach to identify mutations in gram-positive Streptococcus Pneumonia.
The fast and accurate detection of infections with Pseudomo- nas species and the prediction of response to anti-microbial therapy represent a high unmet clinical need.
This need is addressed by the present invention. Summary of the Invention
The present inventors addressed this need by carrying out whole genome sequencing of a large cohort of Pseudomonas clinical isolates and comparing the genetic mutation profile to classical culture based antimicrobial susceptibility test¬ ing with the goal to develop a test which can be used to de¬ tect bacterial susceptibility/resistance against antimicrobi¬ al drugs using molecular testing.
The inventors performed extensive studies on the genome of bacteria of Pseudomonas species either susceptible or re¬ sistant to antimicrobial, e.g. antibiotic, drugs. Based on this information, it is now possible to provide a detailed analysis on the resistance pattern of Pseudomonas strains based on individual genes or mutations on a nucleotide level. This analysis involves the identification of a resistance against individual antimicrobial, e.g. antibiotic, drugs as well as clusters of them. This allows not only for the deter- mination of a resistance to a single antimicrobial, e.g. an¬ tibiotic, drug, but also to groups of antimicrobial drugs, e.g. antibiotics such as lactam or quinolone antibiotics, or even to all relevant antibiotic drugs. Therefore, the present invention will considerably facilitate the selection of an appropriate antimicrobial, e.g. antibi¬ otic, drug for the treatment of a Pseudomonas infection in a patient and thus will largely improve the quality of diagno¬ sis and treatment.
According to a first aspect, the present invention discloses a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicro¬ bial drug treatment, which can be also described as a method of determining an antimicrobial drug, e.g. antibiotic, re¬ sistant Pseudomonas infection of a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 1 or
Table 2 below, wherein the presence of said at least two mu¬ tations is indicative of an infection with an antimicrobial drug resistant, e.g. antibiotic resistant, Pseudomonas strain in said patient.
An infection of a patient with Pseudomonas species potential¬ ly resistant to antimicrobial drug treatment herein means an infection of a patient with Pseudomonas species wherein it is unclear if the Pseudomonas species is susceptible to treat- ment with a specific antimicrobial drug or if it is resistant to the antimicrobial drug.
In step b) above, as well as corresponding steps, at least one mutation in at least two genes is determined, so that in total at least two mutations are determined, wherein the two mutations are in different genes.
Table 1: List of genes
SCV20265_1892 SCV20265_5625 SCV20265_1467 SCV20265_5607 SCV20265_3294
SCV20265_1879 SCV20265_5242 SCV20265_2224 SCV20265_0530 SCV20265_3289
SCV20265_1858 SCV20265_2193 SCV20265_6274 SCV20265_2958 SCV20265_3248
SCV20265_1132 SCV20265_1451 SCV20265_6120 SCV20265_4839 SCV20265_2195
SCV20265_0968 SCV20265_2464 SCV20265_2518 SCV20265_2654 SCV20265_3101
SCV20265_3909 SCV20265_2610 SCV20265_1805 SCV20265_4445 SCV20265_2883
SCV20265_2916 SCV20265_1721 SCV20265_3099 SCV20265_1735 SCV20265_6289
SCV20265_2974 SCV20265_2404 SCV20265_6135 SCV20265_3626 SCV20265_1050
SCV20265_0188 SCV20265_5329 SCV20265_2792 SCV20265_1617 SCV20265_2236
SCV20265_0491 SCV20265_2422 SCV20265_5463 SCV20265_5597 SCV20265_0241 Table 2: List of genes
Figure imgf000009_0001
According to a second aspect, the present invention relates to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, e.g. from an antimicrobial drug, e.g. antibiotic, re¬ sistant Pseudomonas infection, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa- tient;
b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 1 or Table 2 above, wherein the presence of said at least two mu¬ tations is indicative of a resistance to one or more antimi- crobial, e.g. antibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection.
A third aspect of the present invention relates to a method of determining an antimicrobial drug, e.g. antibiotic, re¬ sistance profile for bacterial microorganisms of Pseudomonas species, comprising: obtaining or providing a first data set of gene sequences of a plurality of clinical isolates of Pseudomonas species;
providing a second data set of antimicrobial drug, e.g. anti¬ biotic, resistance of the plurality of clinical isolates of Pseudomonas species;
aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Pseudomonas, and/or assembling the gene sequence of the first data set, at least in part;
analyzing the gene sequences of the first data set for genet¬ ic variants to obtain a third data set of genetic variants; correlating the third data set with the second data set and statistically analyzing the correlation; and
determining the genetic sites in the genome of Pseudomonas associated with antimicrobial drug, e.g. antibiotic, re¬ sistance .
In addition, the present invention relates in a fourth aspect to a method of determining an antimicrobial drug, e.g. anti- biotic, resistance profile for a bacterial microorganism be¬ longing to the species Pseudomonas comprising the steps of a) obtaining or providing a sample containing or suspected of containing the bacterial microorganism;
b) determining the presence of a mutation in at least one gene of the bacterial microorganism as determined by the method according to the third aspect of the present inven¬ tion;
wherein the presence of a mutation is indicative of a re¬ sistance to an antimicrobial, e.g. antibiotic, drug.
Furthermore, the present invention discloses in a fifth as¬ pect a diagnostic method of determining an infection of a pa¬ tient with Pseudomonas species potentially resistant to anti- microbial drug treatment, which can, like in the first as¬ pect, also be described as method of determining an antimi¬ crobial drug, e.g. antibiotic, resistant Pseudomonas infec¬ tion of a patient, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Pseudomonas from the patient;
b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism belonging to the species Pseudomonas as determined by the method according to the third aspect of the present invention, wherein the presence of said at least one mutation is indicative of an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection in said patient.
Also disclosed is in a sixth aspect a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, e.g. from an antimi¬ crobial drug, e.g. antibiotic, resistant Pseudomonas infec- tion, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Pseudomonas from the patient;
b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism belonging to the species Pseudomonas as determined by the method according to the third aspect of the present invention, wherein the presence of said at least one mutation is indicative of a re¬ sistance to one or more antimicrobial, e.g. antibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection. A seventh aspect of the present invention relates to a method of acquiring, respectively determining, an antimicrobial drug, e.g. antibiotic, resistance profile for a bacterial mi¬ croorganisms of Pseudomonas species, comprising:
obtaining or providing a first data set of gene sequences of a clinical isolate of Pseudomonas species;
providing a second data set of antimicrobial drug, e.g. anti¬ biotic, resistance of a plurality of clinical isolates of Pseudomonas species;
aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Pseudomonas, and/or assembling the gene sequence of the first data set, at least in part;
analyzing the gene sequences of the first data set for genet¬ ic variants to obtain a third data set of genetic variants of the first data set;
correlating the third data set with the second data set and statistically analyzing the correlation; and
determining the genetic sites in the genome of Pseudomonas of the first data set associated with antimicrobial drug, e.g. antibiotic, resistance.
According to an eighth aspect, the present invention disclos¬ es a computer program product comprising executable instruc¬ tions which, when executed, perform a method according to the third, fourth, fifth, sixth or seventh aspect of the present invention . Further aspects and embodiments of the invention are dis¬ closed in the dependent claims and can be taken from the fol¬ lowing description, figures and examples, without being limited thereto.
Figures
The enclosed drawings should illustrate embodiments of the present invention and convey a further understanding thereof. In connection with the description they serve as explanation of concepts and principles of the invention. Other embodi¬ ments and many of the stated advantages can be derived in re¬ lation to the drawings. The elements of the drawings are not necessarily to scale towards each other. Identical, function- ally equivalent and acting equal features and components are denoted in the figures of the drawings with the same refer¬ ence numbers, unless noted otherwise.
Fig. 1 shows schematically a read-out concept for a diagnos- tic test according to a method of the present invention.
Detailed description of the present invention
Definitions
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. An "antimicrobial drug" in the present invention refers to a group of drugs that includes antibiotics, antifungals, antiprotozoals, and antivirals. According to certain embodi¬ ments, the antimicrobial drug is an antibiotic. The term "nucleic acid molecule" refers to a polynucleotide molecule having a defined sequence. It comprises DNA mole¬ cules, RNA molecules, nucleotide analog molecules and combi¬ nations and derivatives thereof, such as DNA molecules or RNA molecules with incorporated nucleotide analogs or cDNA.
The term "nucleic acid sequence information" relates to an information which can be derived from the sequence of a nu¬ cleic acid molecule, such as the sequence itself or a varia- tion in the sequence as compared to a reference sequence.
The term "mutation" relates to a variation in the sequence as compared to a reference sequence. Such a reference sequence can be a sequence determined in a predominant wild type or- ganism or a reference organism, e.g. a defined and known bac¬ terial strain or substrain. A mutation is for example a deletion of one or multiple nucleotides, an insertion of one or multiple nucleotides, or substitution of one or multiple nu¬ cleotides, duplication of one or a sequence of multiple nu- cleotides, translocation of one or a sequence of multiple nu¬ cleotides, and, in particular, a single nucleotide polymor¬ phism (SNP) .
In the context of the present invention a "sample" is a sam- pie which comprises at least one nucleic acid molecule from a bacterial microorganism. Examples for samples are: cells, tissue, body fluids, biopsy specimens, blood, urine, saliva, sputum, plasma, serum, cell culture supernatant, swab sample and others. According to certain embodiments, the sample is a patient sample (clinical isolate) .
New and highly efficient methods of sequencing nucleic acids referred to as next generation sequencing have opened the possibility of large scale genomic analysis. The term "next generation sequencing" or "high throughput sequencing" refers to high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once. Examples include Massively Parallel Signa¬ ture Sequencing (MPSS) , Polony sequencing, 454
pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, Helioscope (TM) single molecule sequencing, Single Molecule SMRT(TM) sequencing, Single Molecule real time (RNAP) se¬ quencing, Nanopore DNA sequencing, Sequencing By Hybridization, Amplicon Sequencing, GnuBio.
Within the present description the term "microorganism" com- prises the term microbe. The type of microorganism is not particularly restricted, unless noted otherwise or obvious, and, for example, comprises bacteria, viruses, fungi, micro¬ scopic algae und protozoa, as well as combinations thereof. According to certain aspects, it refers to one or more Pseu- domonas species, particularly Pseudomonas aeruginosa.
A reference to a microorganism or microorganisms in the pre¬ sent description comprises a reference to one microorganism as well a plurality of microorganisms, e.g. two, three, four, five, six or more microorganisms.
A vertebrate within the present invention refers to animals having a vertebrae, which includes mammals - including hu¬ mans, birds, reptiles, amphibians and fishes. The present in- vention thus is not only suitable for human medicine, but al¬ so for veterinary medicine. According to certain embodiments, the patient in the present methods is a vertebrate, more preferably a mammal and most preferred a human patient. Before the invention is described in exemplary detail, it is to be understood that this invention is not limited to the particular component parts of the process steps of the meth¬ ods described herein as such methods may vary. It is also to be understood that the terminology used herein is for purpos- es of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. For example, the term "a" as used herein can be understood as one single entity or in the meaning of "one or more" entities. It is al¬ so to be understood that plural forms include singular and/or plural referents unless the context clearly dictates other¬ wise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.
Regarding the dosage of the antimicrobial, e.g. antibiotic, drugs, it is referred to the established principles of phar- macology in human and veterinary medicine. For example, Forth, Henschler, Rummel "Allgemeine und spezielle
Pharmakologie und Toxikologie" , 9th edition, 2005 might be used as a guideline. Regarding the formulation of a ready-to- use medicament, reference is made to "Remington, The Science and Practice of Pharmacy", 22nd edition, 2013.
Assembling of a gene sequence can be carried out by any known method and is not particularly limited. According to certain embodiments, mutations that were found using alignments can also be compared or matched with align¬ ment-free methods, e.g. for detecting single base exchanges, for example based on contigs that were found by assemblies. For example, reads obtained from sequencing can be assembled to contigs and the contigs can be compared to each other.
According to a first aspect, the present invention relates to a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicro¬ bial drug treatment, which can also be described as method of determining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection of a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least two genes from the group of genes consisting of
SCV20265 1892, SCV20265 _5625 , SCV20265 1467, SCV20265 5607,
SCV20265 3294, SCV20265 1879 , SCV20265 5242, SCV20265 2224,
SCV20265 _0530, SCV20265 _3289 , SCV20265 _1858, SCV20265 2193,
SCV20265 6274, SCV20265 _2958 , SCV20265 3248, SCV20265 1132,
SCV20265 1451, SCV20265 _6120 , SCV20265 _4839, SCV20265 2195,
SCV20265 _0968, SCV20265 2464 , SCV20265 _2518, SCV20265 2654,
SCV20265 3101, SCV20265 _3909 , SCV20265 _2610, SCV20265 1805,
SCV20265 4445, SCV20265 _2883 , SCV20265 2916, SCV20265 1721,
SCV20265 _3099, SCV20265 _1735 , SCV20265 _6289, SCV20265 2974,
SCV20265 2404, SCV20265 _6135 , SCV20265 _3626, SCV20265 1050,
SCV20265 _0188, SCV20265 _5329 , SCV20265 2792, SCV20265 1617,
SCV20265 2236, SCV20265 0491 , SCV20265 2422, SCV20265 5463,
SCV20265 _5597, and SCV20265 0241, wherein the presence of said at least two mutations is indicative of an infection with an antimicrobial, e.g. antibiotic, resistant Pseudomonas strain in said patient.
In this method, as well as the other methods of the inven- tion, the sample can be provided or obtained in any way, preferably non-invasive, and can be e.g. provided as an in vitro sample or prepared as in vitro sample.
According to certain aspects, mutations in at least two, three, four, five, six, seven, eight, nine or ten genes are determined in any of the methods of the present invention, e.g. in at least two genes or in at least three genes. In¬ stead of testing only single genes or mutants, a combination of several variant positions can improve the prediction accu- racy and further reduce false positive findings that are in¬ fluenced by other factors. Therefore, it is in particular preferred to determine the presence of a mutation in 2, 3, 4, 5, 6, 7, 8 or 9 (or more) genes selected from Table 1 or 2. For the above genes, i.e. the genes also denoted in Tables 1 and 2, the highest probability of a resistance to at least one antimicrobial drug, e.g. antibiotic, could be observed, with p-values smaller than 10 , particularly smaller than 10~35, further particularly smaller than 10~40, indicating the high significance of the values (n= 1104; a = 0.05) . Details regarding Tables 1 and 2 can be taken from Tables 3 and 4 (4a, 4b, 4c) disclosed in the Examples. Having at least two genes with mutations determined, a high probability of an an¬ timicrobial drug, e.g. antibiotic, resistance could be deter- mined. The genes in Table 1 thereby represent the 50 best genes for which a mutation was observed in the genomes of Pseudomonas species, whereas the genes in Table 2 represent the 50 best genes for which a cross-correlation could be ob- served for the antimicrobial drug, e.g. antibiotic, suscepti¬ bility testing for Pseudomonas species as described below.
According to certain embodiments, the obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the patient in this method - as well as the other methods of the invention - can comprise the fol¬ lowing :
A sample of a vertebrate, e.g. a human, e.g. is provided or obtained and nucleic acid sequences, e.g. DNA or RNA sequenc¬ es, are recorded by a known method for recording nucleic ac¬ id, which is not particularly limited. For example, nucleic acid can be recorded by a sequencing method, wherein any se¬ quencing method is appropriate, particularly sequencing meth- ods wherein a multitude of sample components, as e.g. in a blood sample, can be analyzed for nucleic acids and/or nucle¬ ic acid fragments and/or parts thereof contained therein in a short period of time, including the nucleic acids and/or nu¬ cleic acid fragments and/or parts thereof of at least one mi- croorganism of interest, particularly of at least one Pseudo¬ monas species. For example, sequencing can be carried out us¬ ing polymerase chain reaction (PCR) , particularly multiplex PCR, or high throughput sequencing or next generation sequencing, preferably using high-throughput sequencing. For sequencing, preferably an in vitro sample is used.
The data obtained by the sequencing can be in any format, and can then be used to identify the nucleic acids, and thus genes, of the microorganism, e.g. of Pseudomonas species, to be identified, by known methods, e.g. fingerprinting methods, comparing genomes and/or aligning to at least one, or more, genomes of one or more species of the microorganism of inter¬ est, i.e. a reference genome, etc., forming a third data set of aligned genes for a Pseudomonas species - discarding addi¬ tional data from other sources, e.g. the vertebrate. Refer¬ ence genomes are not particularly limited and can be taken from several databases. Depending on the microorganism, dif- ferent reference genomes or more than one reference genomes can be used for aligning. Using the reference genome - as well as also the data from the genomes of the other species, e.g. Pseudomonas species - mutations in the genes for each species and for the whole multitude of samples of different species, e.g. Pseudomonas species, can be obtained.
For example, it is useful in genome-wide association studies to reference the points of interest, e.g. mutations, to one constant reference for enhanced standardization. In case of the human with a high consistency of the genome and 99% iden¬ tical sequences among individuals this is easy and represents the standard, as corresponding reference genomes are availa¬ ble in databases. In case of organisms that trigger infec¬ tious diseases (e.g. bacteria and viruses) this is much more difficult, though. One possibility is to fall back on a vir¬ tual pan genome which contains all sequences of a certain ge¬ nus. A further possibility is the analysis of all available references, which is much more complex. Therein all n refer¬ ences from a database (e.g. RefSeq) are extracted and com- pared with the newly sequenced bacterial genomes k. After this, matrices (% of mapped reads, % of covered genome) are applied to estimate which reference is best suited to all new bacteria. However, n x k complete alignments are carried out. Having a big number of references, though, stable results can be obtained, as is the case for Pseudomonas.
According to certain embodiments, the genomes of Pseudomonas species are referenced to one reference genome. However, it is not excluded that for other microorganisms more than one reference genome is used. In the present methods, the refer¬ ence genome of Pseudomonas is NC_023149 as annotated at the NCBI according to certain embodiments. The reference genome is attached to this application as sequence listing.
The reference sequence was obtained from Pseudomonas strain NC_023149 (http : //www . genome . jp/dbget- bin/www_bget?refseq+NC_023149)
LOCUS NC_023149 6725183 bp DNA circular CON 07-FEB-2015 DEFINITION Pseudomonas aeruginosa SCV20265, complete genome. ACCESSION NC_023149
VERSION NC_023149.1 GI:568306739
DBLINK BioProject: PRJNA224116
BioSample: SAMN02415141
Assembly: GCF_000510305.1
KEYWORDS RefSeq.
SOURCE Pseudomonas aeruginosa SCV20265
ORGANISM Pseudomonas aeruginosa SCV20265
Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales ; Pseudomonadaceae ; Pseudomonas.
REFERENCE 1 (bases 1 to 6725183)
AUTHORS Eckweiler, D . , Bunk,B., Sproer,C, Overmann, J. and Haussler, S .
TITLE Complete Genome Sequence of Highly Adherent Pseu¬ domonas aeruginosa Small-Colony Variant SCV20265
JOURNAL Genome Announc 2 (1) (2014)
PUBMED 24459283
REMARK Publication Status: Online-Only
REFERENCE 2 (bases 1 to 6725183)
AUTHORS Eckweiler, D . , Bunk,B., Overmann, J.
Haeussler,
TITLE JOURNAL Submitted (02-DEC-2013) Bioinformatics , Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, Braunschweig 38124, Germany Alternatively or in addition, the gene sequence of the first data set can be assembled, at least in part, with known meth¬ ods, e.g. by de-novo assembly or mapping assembly. The se¬ quence assembly is not particularly limited, and any known genome assembler can be used, e.g. based on Sanger, 454, Solexa, Illumina, SOLid technologies, etc., as well as hy¬ brids/mixtures thereof.
According to certain embodiments, the data of nucleic acids of different origin than the microorganism of interest, e.g. Pseudomonas species, can be removed after the nucleic acids of interest are identified, e.g. by filtering the data out. Such data can e.g. include nucleic acids of the patient, e.g. the vertebrate, e.g. human, and/or other microorganisms, etc. This can be done by e.g. computational subtraction, as devel- oped by Meyerson et al . 2002. For this, also aligning to the genome of the vertebrate, etc., is possible. For aligning, several alignment-tools are available. This way the original data amount from the sample can be drastically reduced. Also after such removal of "excess" data, fingerprinting and/or aligning, and/or assembly, etc. can be carried out, as described above, forming a third data set of aligned and/or assembled genes for a Pseudomonas species. Using these techniques, genes with mutations of the microor¬ ganism of interest, e.g. Pseudomonas species, can be obtained for various species. When testing these same species for antimicrobial drug, e.g. antibiotic, susceptibility of a number of antimicrobial drugs, e.g. antibiotics, e.g. using standard culturing meth¬ ods on dishes with antimicrobial drug, e.g. antibiotic, in- take, as e.g. described below, the results of these antimi¬ crobial drug, e.g. antibiotic, susceptibility tests can then be cross-referenced/correlated with the mutations in the ge¬ nome of the respective microorganism, e.g. Pseudomonas . Using several, e.g. 50 or more than 50, 100 or more than 100, 200 or more than 200, 300 or more than 300, 400 or more than 400, or 500 or more than 500, e.g. 1000 or more than 1000, e.g. 1100 or more than 1100 different species of a microorganism, e.g. different Pseudomonas species, statistical analysis can be carried out on the obtained cross-referenced data between mutations and antimicrobial drug, e.g. antibiotic, suscepti¬ bility for these number of species, using known methods.
Regarding culturing methods, samples can be e.g. cultured overnight. On the next day individual colonies can be used for identification of organisms, either by culturing or using mass spectroscopy. Based on the identity of organisms new plates containing increasing concentration of antibiotics used for the treatment of these organisms are inoculated and grown for additional 12 - 24 hours. The lowest drug concen- tration which inhibits growth (minimal inhibitory concentra¬ tion - MIC) can be used to determine susceptibil¬ ity/resistance for tested antibiotics.
Correlation of the nucleic acid / gene mutations with antimi- crobial drug, e.g. antibiotic, resistance can be carried out in a usual way and is not particularly limited. For example, resistances can be correlated to certain genes or certain mu¬ tations, e.g. SNPs, in genes. After correlation, statistical analysis can be carried out. In addition, statistical analysis of the correlation of the gene mutations with antimicrobial drug, e.g. antibiotic, re¬ sistance is not particularly limited and can be carried out, depending on e.g. the amount of data, in different ways, for example using analysis of variance (ANOVA) or Student's t- test, for example with a sample size n of 50, 100, 200, 300, 400, 500, e.g. 1000 or 1100, and a level of significance ( - error-level) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller. A statistical value can be obtained for each gene and/or each position in the genome as well as for all antibiotics tested, a group of antibiotics or a single anti¬ biotic. The obtained p-values can also be adapted for statis¬ tical errors, if needed. For statistically sound results a multitude of individuals should be sampled, with n = 50, 100, 200, 300, 400 or 500, e.g. 1000 or 1100, and a level of significance ( -error- level) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller. According to certain embodiments, particularly sig- nificant results can be obtained for n = 200, 200, 400 or 500, e.g. 1000 or 1100.
For statistically sound results a multitude of individuals should be sampled, with n = 50 or more, 100 or more, 200 or more, 300 or more, 400 or more or 500 or more, e.g. 1000 or more or 1100 or more, and a level of significance ( -error- level) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller. According to certain embodiments, particularly significant results can be obtained for n = 200 or more, 200 or more, 400 or more or 500 or more, e.g. 1000 or more or 1100 or more. After the above procedure has been carried out for more than 1100, e.g. 1104, individual species of Pseudomonas, the data disclosed in Tables 1 and 2 were obtained for the statisti¬ cally best correlations between gene mutations and antimicro¬ bial drug, e.g. antibiotic, resistances. Thus, mutations in these genes were proven as valid markers for antimicrobial drug, e.g. antibiotic, resistance.
According to a further aspect, the present invention relates in a second aspect to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, e.g. from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least two genes from the group of genes consisting of
SCV20265 1892, SCV20265 _5625 , SCV20265 1467, SCV20265 5607,
SCV20265 3294, SCV20265 1879 , SCV20265 5242, SCV20265 2224,
SCV20265 _0530, SCV20265 _3289 , SCV20265 _1858, SCV20265 2193,
SCV20265 6274, SCV20265 _2958 , SCV20265 3248, SCV20265 1132,
SCV20265 1451, SCV20265 _6120 , SCV20265 _4839, SCV20265 2195,
SCV20265 _0968, SCV20265 2464 , SCV20265 _2518, SCV20265 2654,
SCV20265 3101, SCV20265 _3909 , SCV20265 _2610, SCV20265 1805,
SCV20265 4445, SCV20265 _2883 , SCV20265 2916, SCV20265 1721,
SCV20265 _3099, SCV20265 _1735 , SCV20265 _6289, SCV20265 2974,
SCV20265 2404, SCV20265 _6135 , SCV20265 _3626, SCV20265 1050,
SCV20265 _0188, SCV20265 _5329 , SCV20265 2792, SCV20265 1617,
SCV20265 2236, SCV20265 0491 , SCV20265 2422, SCV20265 5463,
SCV20265 _5597, and SCV20265 0241, wherein the presence of said at least two mutations is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection.
In this method, the steps a) of obtaining or providing a sam- pie and b) of determining the presence of at least one muta¬ tion are as in the method of the first aspect.
The identification of the at least one or more antimicrobial, e.g. antibiotic, drug in step c) is then based on the results obtained in step b) and corresponds to the antimicrobial, e.g. antibiotic, drug(s) that correlate (s) with the muta¬ tions. Once these antimicrobial drugs, e.g. antibiotics, are ruled out, the remaining antimicrobial drugs, e.g. antibiotic drugs/antibiotics, can be selected in step d) as being suita- ble for treatment.
In the description, references to the first and second aspect also apply to the 14th, 15th, 16th and 17th aspect, referring to the same genes, unless clear from the context that they don't apply.
According to certain embodiments in the method of the first or second aspect, at least a mutation in SCV20265_1892 and/or SCV20265_5625, particularly in positions 1979239 and/or
5987559, respectively, with regard to reference genome
NC_023149 as annotated at the NCBI, is determined. For such mutations, a particularly relevant correlation with antimicrobial drug, e.g. antibiotic, resistance could be deter- mined. In particular, the mutation in position 1979239 with regard to reference genome NC_023149 as annotated at the NCBI is a non-synonymous coding, particularly a codon change aCc/aTc; aCc/aAc, and the mutation in position 5987559 with re- gard to reference genome NC_023149 as annotated at the NCBI is a non-synonymous coding, particularly a codon change tCg/tTg; tCg/tGg.
According to certain embodiments, the antimicrobial drug, e.g. antibiotic, in the method of the first or second aspect, as well as in the other methods of the invention, is at least one selected from the group of β-lactams, β-lactam inhibi¬ tors, quinolines and derivatives thereof, aminoglycosides, polyketides, respectively tetracyclines, and folate synthesis inhibitors.
In the methods of the invention the resistance of Pseudomonas to one or more antimicrobial, e.g. antibiotic, drugs can be determined according to certain embodiments.
According to certain embodiments, the antimicrobial drug is an antibiotic/antibiotic drug.
According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from lactam antibiotics, and the presence of a mutation in the following genes is determined:
SCV20265_1892, SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_1879, SCV20265_5242 , SCV20265_2224 , SCV20265_0530 , SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958, SCV20265_3248, SCV20265_1451 , SCV20265_6120 , SCV20265_4839, SCV20265_2195 , SCV20265_0968 , SCV20265_2464, SCV20265 2518, SCV20265 2654, SCV20265 3101, SCV20265 1805, SCV20265_4445, SCV20265_2883 , SCV20265_1721 , SCV20265_3099 , SCV20265_1735, SCV20265_6289 , SCV20265_2974 , SCV20265_6135 , SCV20265_3626, SCV20265_1050, SCV20265_5329 , SCV20265_2792 , and/or SCV20265_2236.
According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from quinolone antibiotics, e.g.
fluoroquinolone antibiotics, and the presence of a mutation in the following genes is determined: SCV20265_1892 ,
SCV20265 _5625, SCV20265 1467, SCV20265 _5607, SCV20265 _3294,
SCV20265 1879, SCV20265 5242, SCV20265 2224, SCV20265 _0530,
SCV20265 _3289, SCV20265 _1858, SCV20265 2193, SCV20265 _6274,
SCV20265 _2958, SCV20265 3248, SCV20265 1132, SCV20265 _1451,
SCV20265 _6120, SCV20265 _4839, SCV20265 _2195, SCV20265 _0968,
SCV20265 2464, SCV20265 _2518, SCV20265 _2654, SCV20265 _3101,
SCV20265 _3909, SCV20265 _2610, SCV20265 _1805, SCV20265 4445,
SCV20265 _2883, SCV20265 2916, SCV20265 1721, SCV20265 _3099,
SCV20265 _1735, SCV20265 _6289, SCV20265 2974, SCV20265 2404,
SCV20265 _6135, SCV20265 _3626, SCV20265 _1050, SCV20265 _0188,
SCV20265 _5329, SCV20265 2792, SCV20265 1617, SCV20265 _2236,
SCV20265 0491, SCV20265 2422, SCV20265 _5463, SCV20265 _5597, and/or SCV20265 0241. According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from aminoglycoside antibiotics, and the presence of a mutation in the following genes is determined: SCV20265_1892, SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_3294, SCV20265_1879 , SCV20265_5242 , SCV20265_2224 , SCV20265_0530, SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958 , SCV20265_3248 , SCV20265_1132 , SCV20265 1451, SCV20265 6120, SCV20265 4839, SCV20265 2195, SCV20265_0968, SCV20265_2464, SCV20265_2518, SCV20265_2654, SCV20265_3101, SCV20265_3909, SCV20265_2610, SCV20265_1805, SCV20265_4445, SCV20265_2883, SCV20265_2916, SCV20265_1721, SCV20265_3099, SCV20265_1735, SCV20265_6289, SCV20265_2974, SCV20265_2404, SCV20265_6135, SCV20265_3626, SCV20265_1050, SCV20265_0188, SCV20265_5329, SCV20265_2792, SCV20265_1617, SCV20265_2236, SCV20265_0491, SCV20265_2422, SCV20265 5463, SCV20265 5597, and/or SCV2026 0241.
According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from other antibiotics ( (benzene de¬ rived) /sulfonamide) , and the presence of a mutation in the following genes is determined: SCV20265_1892 , SCV20265_5625,
SCV20265 1467, SCV20265 _5607, SCV20265 3294, SCV20265 _1879,
SCV20265 5242, SCV20265 2224, SCV20265 _0530, SCV20265 _3289,
SCV20265 _1858, SCV20265 2193, SCV20265 6274, SCV20265 _2958,
SCV20265 3248, SCV20265 1132, SCV20265 1451, SCV20265 _6120,
SCV20265 _4839, SCV20265 _2195, SCV20265 _0968, SCV20265 2464,
SCV20265 _2518, SCV20265 _2654, SCV20265 3101, SCV20265 _3909,
SCV20265 _2610, SCV20265 _1805, SCV20265 4445, SCV20265 _2883,
SCV20265 2916, SCV20265 1721, SCV20265 _3099, SCV20265 _1735,
SCV20265 _6289, SCV20265 2974, SCV20265 2404, SCV20265 _6135,
SCV20265 _3626, SCV20265 _1050, SCV20265 _0188, SCV20265 _5329,
SCV20265 2792, SCV20265 1617, SCV20265 2236, SCV20265 _0491,
SCV20265 2422, SCV20265 _5463, SCV20265 _5597, and/or
SCV20265 0241.
According to certain embodiments of the first and/or second aspect of the invention, determining the nucleic acid se¬ quence information or the presence of a mutation comprises determining the presence of a single nucleotide at a single position in a gene. Thus the invention comprises methods wherein the presence of a single nucleotide polymorphism or mutation at a single nucleotide position is detected.
According to certain embodiments, the antibiotic drug in the methods of the present invention is selected from the group consisting of Amoxicillin/K Clavulanate (AUG) , Ampicillin (AM), Aztreonam (AZT) , Cefazolin (CFZ) , Cefepime (CPE),
Cefotaxime (CFT) , Ceftazidime (CAZ) , Ceftriaxone (CAX) , Ce- furoxime (CRM) , Cephalotin (CF) , Ciprofloxacin (CP) ,
Ertapenem (ETP) , Gentamicin (GM) , Imipenem (IMP), Levofloxa- cin (LVX) , Meropenem (MER) , Piperacillin/Tazobactam (P/T) , Ampicillin/Sulbactam (A/S), Tetracycline (TE) , Tobramycin (TO), and Trimethoprim/Sulfamethoxazole (T/S). The inventors have surprisingly found that mutations in cer¬ tain genes are indicative not only for a resistance to one single antimicrobial, e.g. antibiotic, drug, but to groups containing several drugs . According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from lactam antibiotics and a mutation in at least one of the following genes is detected with regard to reference genome NC 023149: SCV20265 _1892,
SCV20265 _5625, SCV20265 _1467, SCV20265 _5607, SCV20265 _1879,
SCV20265 5242, SCV20265 2224, SCV20265 _0530, SCV20265 _3289,
SCV20265 _1858, SCV20265 _2193, SCV20265 _6274, SCV20265 _2958,
SCV20265 _3248, SCV20265 _1451, SCV20265 _6120, SCV20265 _4839,
SCV20265 _2195, SCV20265 _0968, SCV20265 2464, SCV20265 _2518,
SCV20265 _2654, SCV20265 _3101, SCV20265 _1805, SCV20265 4445,
SCV20265 _2883, SCV20265 1721, SCV20265 _3099, SCV20265 _1735,
SCV20265 _6289, SCV20265 2974, SCV20265 _6135, SCV20265 _3626,
SCV20265 1050, SCV20265 5329, SCV20265 2792, SCV20265 2236. According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from quinolone antibiotics, e.g. fluoroquinolone antibiotics, and a mutation in at least one of the following genes is detected with regard to refer¬ ence genome NC_023149: SCV20265_1892 , SCV20265_5625,
SCV20265 1467, SCV20265 _5607, SCV20265 3294, SCV20265 _1879,
SCV20265 5242, SCV20265 2224, SCV20265 _0530, SCV20265 _3289,
SCV20265 _1858, SCV20265 2193, SCV20265 6274, SCV20265 _2958,
SCV20265 3248, SCV20265 1132, SCV20265 1451, SCV20265 _6120,
SCV20265 _4839, SCV20265 _2195, SCV20265 _0968, SCV20265 2464,
SCV20265 _2518, SCV20265 _2654, SCV20265 3101, SCV20265 _3909,
SCV20265 _2610, SCV20265 _1805, SCV20265 4445, SCV20265 _2883,
SCV20265 2916, SCV20265 1721, SCV20265 _3099, SCV20265 _1735,
SCV20265 _6289, SCV20265 2974, SCV20265 2404, SCV20265 _6135,
SCV20265 _3626, SCV20265 _1050, SCV20265 _0188, SCV20265 _5329,
SCV20265 2792, SCV20265 1617, SCV20265 2236, SCV20265 _0491,
SCV20265 2422, SCV20265 5463, SCV20265 5597, SCV20265 0241.
According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from aminoglycoside antibiot¬ ics and a mutation in at least one of the following genes is detected with regard to reference genome NC 023149:
SCV20265 _1892, SCV20265 _5625, SCV20265 _1467, SCV20265 _5607,
SCV20265 _3294, SCV20265 _1879, SCV20265 5242, SCV20265 2224,
SCV20265 _0530, SCV20265 _3289, SCV20265 _1858, SCV20265 _2193,
SCV20265 _6274, SCV20265 _2958, SCV20265 _3248, SCV20265 1132,
SCV20265 _1451, SCV20265 _6120, SCV20265 _4839, SCV20265 _2195,
SCV20265 _0968, SCV20265 2464, SCV20265 _2518, SCV20265 _2654,
SCV20265 _3101, SCV20265 _3909, SCV20265 _2610, SCV20265 _1805,
SCV20265 4445, SCV20265 _2883, SCV20265 _2916, SCV20265 1721,
SCV20265 3099, SCV20265 1735, SCV20265 6289, SCV20265 2974, SCV20265_2404, SCV20265_6135 , SCV20265_3626 , SCV20265_1050 , SCV20265_0188, SCV20265_5329, SCV20265_2792, SCV20265_1617 , SCV20265_2236, SCV20265_0491 , SCV20265_2422 , SCV20265_5463 , SCV20265_5597, SCV20265_0241.
According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from other antibiotics ( (ben¬ zene derived) /sulfonamide) and a mutation in at least one of the following genes is detected with regard to reference ge- nome NC 023149 : SCV20265 _1892, SCV20265 _5625, SCV20265 1467
SCV20265 _5607, SCV20265 3294, SCV20265 1879, SCV20265 5242,
SCV20265 2224, SCV20265 0530, SCV20265 3289, SCV20265 1858,
SCV20265 2193, SCV20265 6274, SCV20265 2958, SCV20265 3248,
SCV20265 1132, SCV20265 1451, SCV20265 6120, SCV20265 4839,
SCV20265 _2195, SCV20265 0968, SCV20265 2464, SCV20265 2518,
SCV20265 _2654, SCV20265 3101, SCV20265 3909, SCV20265 2610,
SCV20265 _1805, SCV20265 4445, SCV20265 2883, SCV20265 2916,
SCV20265 1721, SCV20265 3099, SCV20265 1735, SCV20265 6289,
SCV20265 2974, SCV20265 2404, SCV20265 6135, SCV20265 3626,
SCV20265 _1050, SCV20265 0188, SCV20265 5329, SCV20265 2792,
SCV20265 1617, SCV20265 2236, SCV20265 0491, SCV20265 2422,
SCV20265 _5463, SCV20265 5597, SCV20265 0241. For specific antimicrobial drugs, e.g. antibiotics, specific positions in the above genes can be determined where a high statistical significance is observed. The inventors found that, apart from the above genes indicative of a resistance against antibiotics, also single nucleotide polymorphisms (= SNP's) may have a high significance for the presence of a re¬ sistance against defined antibiotic drugs. The analysis of these polymorphisms on a nucleotide level may further improve and accelerate the determination of a drug resistance to an¬ timicrobial drugs, e.g. antibiotics, in Pseudomonas .
According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from lactam antibiotics and a mutation in at least one of the following nucleotide posi¬ tions is detected with regard to reference genome NC_023149: 1979239, 5987559, 1537406, 5965080, 1967346, 5569783 2350860, 562872, 3507580, 1947689, 2316386, 6685845, 3142437 3468647, 1521674, 6520799, 5124971, 2317909, 1009933 2567532, 2611669, 2754829, 3301233, 1899865, 4712288 3019764, 1805165, 3299685, 1821163, 6702956, 3160788 6535290, 3881624, 1099519, 5662982, 2903129, 2363393 2350862, 3507601, 3507667, 6519971
According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from quinolone antibiotics, e.g. fluoroquinolone antibiotics, and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149: 1979239, 5987559,
1537406, 5965080, 3513162, 1967346, 5569783, 2350860, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647,
1194383, 1521674, 6520799, 5124971, 2317909, 1009933,
2567532, 2611669, 2754829, 3301233, 4166792, 2709322,
1899865, 4712288, 3019764, 3068671, 1805165, 3299685,
1821163, 6702956, 3160788, 2515627, 6535290, 3881624,
1099519, 208902, 5662982, 2903129, 1701758, 2363393, 525701, 2530203, 5806684, 5954547, 261978, 2350862, 3507601, 3507667, 6519971. According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from aminoglycoside antibiot¬ ics and a mutation in at least one of the following nucleo- tide positions is detected with regard to reference genome NC_023149: 1979239, 5987559, 1537406, 5965080, 3513162,
1967346, 5569783, 2350860, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647, 1194383, 1521674, 6520799,
5124971, 2317909, 1009933, 2567532, 2611669, 2754829,
3301233, 4166792, 2709322, 1899865, 4712288, 3019764,
3068671, 1805165, 3299685, 1821163, 6702956, 3160788,
2515627, 6535290, 3881624, 1099519, 208902, 5662982, 2903129, 1701758, 2363393, 525701, 2530203, 5806684, 5954547, 261978, 2350862, 3507601, 3507667, 6519971.
According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from other antibiotics ( (ben¬ zene derived) /sulfonamide) and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149: 1979239, 5987559, 1537406,
5965080, 3513162, 1967346, 5569783, 2350860, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647, 1194383,
1521674, 6520799, 5124971, 2317909, 1009933, 2567532,
2611669, 2754829, 3301233, 4166792, 2709322, 1899865,
4712288, 3019764, 3068671, 1805165, 3299685, 1821163,
6702956, 3160788, 2515627, 6535290, 3881624, 1099519, 208902, 5662982, 2903129, 1701758, 2363393, 525701, 2530203, 5806684, 5954547, 261978, 2350862, 3507601, 3507667, 6519971.
According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is T/S, CP, LVX, GM, and/or TO and a mutation in at least one of the following nucleotide positions is detected with regard to reference ge¬ nome NC_023149: 1979239, 5987559, 1537406, 5965080, 3513162, 1967346, 5569783, 2350860, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647, 1194383, 1521674, 6520799,
5124971, 2317909, 1009933, 2567532, 2611669, 2754829,
3301233, 4166792, 2709322, 1899865, 4712288, 3019764,
3068671, 1805165, 3299685, 1821163, 6702956, 3160788,
2515627, 6535290, 3881624, 1099519, 208902, 5662982, 2903129, 1701758, 2363393, 525701, 2530203, 5806684, 5954547, 261978, 2350862, 3507601, 3507667, 6519971.
According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is CPE and a mu¬ tation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149:
1979239, 5987559, 1537406, 5569783, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647, 1521674, 6520799,
5124971, 2317909, 1009933, 2567532, 2611669, 2754829,
1899865, 4712288, 1805165, 1821163, 6702956, 3160788,
6535290, 3881624, 1099519, 5662982, 2903129, 2363393,
3507601, 3507667, 6519971.
According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is P/T and a mu tation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149:
1979239, 5987559, 1537406, 5965080, 1967346, 2350860,
2754829, 3301233, 3019764, 6702956, 3881624, 2350862.
According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is ETP and a mu tation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149:
1979239, 5987559, 2754829, 3301233, 3299685, 1099519.
According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is CFT, IMP, MER, CAX, AZT, and/or CAZ and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149: 1979239, 5987559. According to certain embodiments of the first and/or second aspect of the invention, the resistance of a bacterial micro¬ organism belonging to the species Pseudomonas against 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, 17, 18, 19, 20 or 21 antibiotic drugs is determined.
According to certain embodiments of the first and/or second aspect of the invention, a detected mutation is a mutation leading to an altered amino acid sequence in a polypeptide derived from a respective gene in which the detected mutation is located. According to this aspect, the detected mutation thus leads to a truncated version of the polypeptide (wherein a new stop codon is created by the mutation) or a mutated version of the polypeptide having an amino acid exchange at the respective position.
According to certain embodiments of the first and/or second aspect of the invention, determining the nucleic acid se¬ quence information or the presence of a mutation comprises determining a partial sequence or an entire sequence of the at least two genes.
According to certain embodiments of the first and/or second aspect of the invention, determining the nucleic acid se- quence information or the presence of a mutation comprises determining a partial or entire sequence of the genome of the Pseudomonas species, wherein said partial or entire sequence of the genome comprises at least a partial sequence of said at least two genes.
According to certain embodiments of the first and/or second aspect of the invention, determining the nucleic acid se¬ quence information or the presence of a mutation comprises using a next generation sequencing or high throughput sequencing method. According to preferred embodiments of the first and/or second aspect of the invention, a partial or en¬ tire genome sequence of the bacterial organism of Pseudomonas species is determined by using a next generation sequencing or high throughput sequencing method.
In a further, third aspect, the present invention relates to a method of determining an antimicrobial drug, e.g. antibi¬ otic, resistance profile for bacterial microorganisms of Pseudomonas species, comprising:
obtaining or providing a first data set of gene sequences of a plurality of clinical isolates of Pseudomonas species;
providing a second data set of antimicrobial drug, e.g. anti¬ biotic, resistance of the plurality of clinical isolates of Pseudomonas species;
aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Pseudomonas, and/or assembling the gene sequence of the first data set, at least in part;
analyzing the gene sequences of the first data set for genet¬ ic variants to obtain a third data set of genetic variants; correlating the third data set with the second data set and statistically analyzing the correlation; and determining the genetic sites in the genome of Pseudomonas associated with antimicrobial drug, e.g. antibiotic, re¬ sistance . The different steps can be carried out as described with re¬ gard to the method of the first aspect of the present inven¬ tion .
When referring to the second data set, wherein the second da- ta set e.g. comprises, respectively is, a set of antimicrobi¬ al drug, e.g. antibiotic, resistances of a plurality of clin¬ ical isolates, this can, within the scope of the invention, also refer to a self-learning data base that, whenever a new sample is analyzed, can take this sample into the second data set and thus expand its data base. The second data set thus does not have to be static and can be expanded, either by ex¬ ternal input or by incorporating new data due to self- learning. This is, however, not restricted to the third as¬ pect of the invention, but applies to other aspects of the invention that refer to a second data set, which does not necessarily have to refer to antimicrobial drug resistance. The same applies, where applicable, to the first data set, e.g. in the third aspect. According to certain embodiments, statistical analysis in the present methods is carried out using Fisher' s test with p < 10~6, preferably p < 10~9, particularly p < 10~10.
The method of the third aspect of the present invention, as well as related methods, e.g. according to the 7th and 10th aspect, can, according to certain embodiments, comprise cor¬ relating different genetic sites to each other. This way even higher statistical significance can be achieved. According to certain embodiments of the method of the third aspect and related methods - as above, the second data set is provided by culturing the clinical isolates of Pseudomonas species on agar plates provided with antimicrobial drugs, e.g. antibiotics, at different concentrations and the second data is obtained by taking the minimal concentration of the plates that inhibits growth of the respective Pseudomonas species .
According to certain embodiments of the method of the third aspect and related methods, the antibiotic is at least one selected from the group of β-lactams, β-lactam inhibitors, quinolines and derivatives thereof, aminoglycosides,
tetracyclines, and folate synthesis inhibitors, preferably
Amoxicillin/K Clavulanate, Ampicillin, Aztreonam, Cefazolin, Cefepime, Cefotaxime, Ceftazidime, Ceftriaxone, Cefuroxime, Cephalothin, Ciprofloxacin, Ertapenem, Gentamicin, Imipenem, Levofloxacin, Meropenem, Piperacillin/Tazobactam, Ampicil- lin/Sulbactam, Tetracycline, Tobramycin, and Trimethoprim/Sulfamethoxazole .
According to certain embodiments of the method of the third aspect and related methods, the gene sequences in the third data set are comprised in at least one gene from the group of genes consisting of SCV20265_1892 , SCV20265_5625,
SCV20265_1467, SCV20265_5607 , SCV20265_3294 , SCV20265_1879 , SCV20265_5242, SCV20265_2224 , SCV20265_0530 , SCV20265_3289 , SCV20265_1858, SCV20265_2193 , SCV20265_6274 , SCV20265_2958 , SCV20265_3248, SCV20265_1132 , SCV20265_1451 , SCV20265_6120 , SCV20265_4839, SCV20265_2195 , SCV20265_0968 , SCV20265_2464, SCV20265_2518, SCV20265_2654 , SCV20265_3101 , SCV20265_3909 , SCV20265 2610, SCV20265 1805, SCV20265 4445, SCV20265 2883, SCV20265_2916, SCV20265_1721 , SCV20265_3099 , SCV20265_1735 ,
SCV20265_6289, SCV20265_2974, SCV20265_2404 , SCV20265_6135 ,
SCV20265_3626, SCV20265_1050, SCV20265_0188 , SCV20265_5329 ,
SCV20265_2792, SCV20265_1617 , SCV20265_2236 , SCV20265_0491 ,
SCV20265_2422, SCV20265_5463 , SCV20265_5597 , and
SCV20265 0241, or from the genes listed in Table 5.
According to certain embodiments of the method of the third aspect and related methods, the genetic variant has a point mutation, an insertion and or deletion of up to four bases, and/or a frameshift mutation, particularly a non-synonymous coding in YP 008980900.1 and/or YP 008984625.1.
A fourth aspect of the present invention relates to a method of determining an antimicrobial drug, e.g. antibiotic, re¬ sistance profile for a bacterial microorganism belonging to the species Pseudomonas comprising the steps of
a) obtaining or providing a sample containing or suspected of containing the bacterial microorganism;
b) determining the presence of a mutation in at least one gene of the bacterial microorganism as determined by the method of the third aspect of the invention;
wherein the presence of a mutation is indicative of a re¬ sistance to an antimicrobial drug, e.g. antibiotic, drug.
Steps a) and b) can herein be carried out as described with regard to the first aspect, as well as for the following as¬ pects of the invention.
With this method, any mutations in the genome of Pseudomonas species correlated with antimicrobial drug, e.g. antibiotic, resistance can be determined and a thorough antimicrobial drug, e.g. antibiotic, resistance profile can be established A simple read out concept for a diagnostic test as described in this aspect is shown schematically in Fig. 1.
According to Fig. 1, a sample 1, e.g. blood from a patient, is used for molecular testing 2, e.g. using next generation sequencing (NGS) , and then a molecular fingerprint 3 is taken, e.g. in case of NGS a sequence of selected ge- nomic/plasmid regions or the whole genome is assembled. This is then compared to a reference library 4, i.e. selected se- quences or the whole sequence are/is compared to one or more reference sequences, and mutations (SNPs, sequence- gene ad¬ ditions/deletions, etc.) are correlated with susceptibility/ reference profile of reference strains in the reference li¬ brary. The reference library 4 herein contains many genomes and is different from a reference genome. Then the result 5 is reported comprising ID (pathogen identification), i.e. a list of all (pathogenic) species identified in the sample, and AST (antimicrobial susceptibility testing), i.e. a list including a susceptibility /resistance profile for all spe- cies listed
A fifth aspect of the present invention relates to a diagnos¬ tic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicrobial drug treatment, which also can be described as method of de¬ termining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection in a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the spe- cies Pseudomonas from the patient;
b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism belonging to the species Pseudomonas as determined by the method of the third aspect of the present invention, wherein the presence of said at least one mutation is indicative of an antimicro¬ bial drug, e.g. antibiotic, resistant Pseudomonas infection in said patient.
Again, steps a) and b) can herein be carried out as described with regard to the first aspect of the present invention.
According to this aspect, a Pseudomonas infection in a pa- tient can be determined using sequencing methods as well as a resistance to antimicrobial drugs, e.g. antibiotics, of the Pseudomonas species be determined in a short amount of time compared to the conventional methods. In a sixth aspect the present invention relates to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomonas strain, e.g. an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Pseudomonas from the patient;
b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism belonging to the species Pseudomonas as determined by the method of the third aspect of the invention, wherein the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection. This method can be carried out similarly to the second aspect of the invention and enables a fast was to select a suitable treatment with antibiotics for any infection with an unknown Pseudomonas species.
A seventh aspect of the present invention relates to a method of acquiring, respectively determining, an antimicrobial drug, e.g. antibiotic, resistance profile for a bacterial mi¬ croorganisms of Pseudomonas species, comprising:
obtaining or providing a first data set of gene sequences of a clinical isolate of Pseudomonas species;
providing a second data set of antimicrobial drug, e.g. anti¬ biotic, resistance of a plurality of clinical isolates of Pseudomonas species;
aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Pseudomonas, and/or assembling the gene sequence of the first data set, at least in part;
analyzing the gene sequences of the first data set for genet- ic variants to obtain a third data set of genetic variants of the first data set;
correlating the third data set with the second data set and statistically analyzing the correlation; and
determining the genetic sites in the genome of Pseudomonas of the first data set associated with antimicrobial drug, e.g. antibiotic, resistance.
With this method, antimicrobial drug, e.g. antibiotic, re¬ sistances in an unknown isolate of Pseudomonas can be deter- mined.
According to certain embodiments, the reference genome of Pseudomonas is NC_023149 as annotated at the NCBI . According to certain embodiments, statistical analysis in the present methods is carried out using Fisher's test with p < 10~6, preferably p < 10~9, particularly p < 10~10. Also, according to certain embodiments, the method further comprises corre- lating different genetic sites to each other.
An eighth aspect of the present invention relates to a com¬ puter program product comprising computer executable instructions which, when executed, perform a method according to the third, fourth, fifth, sixth or seventh aspect of the present invention .
In certain embodiments the computer program product is one on which program commands or program codes of a computer program for executing said method are stored. According to certain embodiments the computer program product is a storage medium. The same applies to the computer program products of the as¬ pects mentioned afterwards, i.e. the eleventh aspect of the present invention. As noted above, the computer program prod- ucts of the present invention can be self-learning, e.g. with respect to the first and second data sets.
In order to obtain the best possible information from the highly complex genetic data and develop an optimum model for diagnostic and therapeutical uses as well as the methods of the present invention - which can be applied stably in clinical routine - a thorough in silico analysis can be necessary. The proposed principle is based on a combination of different approaches, e.g. alignment with at least one, preferably more reference genomes and/or assembly of the genome and correla¬ tion of mutations found in every sample, e.g. from each pa¬ tient, with all references and drugs, e.g. antibiotics, and search for mutations which occur in several drug and several strains .
Using the above steps a list of mutations as well of genes is generated. These can be stored in databases and statistical models can be derived from the databases. The statistical models can be based on at least one or more mutations at least one or more genes. Statistical models that can be trained can be combined from mutations and genes. Examples of algorithms that can produce such models are association
Rules, Support Vector Machines, Decision Trees, Decision For¬ ests, Discriminant-Analysis, Cluster-Methods, and many more.
The goal of the training is to allow a reproducible, stand- ardized application during routine procedures.
For this, for example, a genome or parts of the genome of a microorganism can be sequenced from a patient to be diag¬ nosed. Afterwards, core characteristics can be derived from the sequence data which can be used to predict resistance. These are the points in the database used for the final mod¬ el, i.e. at least one mutation or at least one gene, but also combinations of mutations, etc. The corresponding characteristics can be used as input for the statistical model and thus enable a prognosis for new pa¬ tients. Not only the information regarding all resistances of all microorganisms, e.g. of Pseudomonas species, against all drugs, e.g. antibiotics, can be integrated in a computer de- cision support tool, but also corresponding directives (e.g. EUCAST) so that only treatment proposals are made that are in line with the directives. A ninth aspect of the present invention relates to the use of the computer program product according to the eighth aspect for acquiring an antimicrobial drug, e.g. antibiotic, re¬ sistance profile for bacterial microorganisms of Pseudomonas species or in a method of the third aspect of the invention.
In a tenth aspect a method of selecting a treatment of a pa¬ tient having an infection with a bacterial microorganism of Pseudomonas species, comprising:
obtaining or providing a first data set comprising a gene sequence of at least one clinical isolate of the microorganism from the patient;
providing a second data set of antimicrobial drug, e.g. anti¬ biotic, resistance of a plurality of clinical isolates of the microorganism;
aligning the gene sequences of the first data set to at least one, preferably one, reference genome of the microorganism, and/or assembling the gene sequence of the first data set, at least in part;
analyzing the gene sequences of the first data set for genet¬ ic variants to obtain a third data set of genetic variants of the first data set;
correlating the third data set with the second data set of antimicrobial drug, e.g. antibiotic, resistance of a plurali- ty of clinical isolates of the microorganism and statistical¬ ly analyzing the correlation;
determining the genetic sites in the genome of the clinical isolate of the microorganism of the first data set associated with antimicrobial drug, e.g. antibiotic, resistance; and selecting a treatment of the patient with one or more antimi¬ crobial, e.g. antibiotic, drugs different from the ones iden¬ tified in the determination of the genetic sites associated with antimicrobial drug, e.g. antibiotic, resistance is dis¬ closed .
Again, the steps can be carried out as similar steps before. In this method, as well as similar ones, no aligning is nec¬ essary, as the unknown sample can be directly correlated, af¬ ter the genome or genome sequences are produced, with the se¬ cond data set and thus mutations and antimicrobial drug, e.g. antibiotic, resistances can be determined. The first data set can be assembled, for example, using known techniques.
According to certain embodiments, statistical analysis in the present method is carried out using Fisher' s test with p < 10~6, preferably p < 10~9, particularly p < 10~10. Also, ac- cording to certain embodiments, the method further comprises correlating different genetic sites to each other.
An eleventh aspect of the present invention is directed to a computer program product comprising computer executable in- structions which, when executed, perform a method according to the tenth aspect.
According to a twelfth aspect of the present invention, a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicrobial drug treatment, which can also be described as a method of determining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection of a patient is disclosed, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 5, wherein the presence of said at least two mutations is indic¬ ative of an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection in said patient.
A thirteenth aspect of the invention discloses a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infec- tion, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 5, wherein the presence of said at least two mutations is indic¬ ative of a resistance to one or more antimicrobial, e.g. an¬ tibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection. Again, the steps can be carried out as in similar methods be¬ fore, e.g. as in the first and second aspect of the inven¬ tion. In the twelfth and thirteenth aspect of the invention, all classes of antibiotics considered in the present method are covered.
Herein, the genes in Table 5 are the following:
SCV20265_1892, SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265 3294, SCV20265 1879, SCV20265 5242, SCV20265 2224, SCV20265 _0530, SCV20265 _3289, SCV20265 _1858, SCV20265 _2193,
SCV20265 6274, SCV20265 _2958, SCV20265 3248, SCV20265 1132,
SCV20265 1451, SCV20265 _6120, SCV20265 _4839, SCV20265 _2195,
SCV20265 _0968, SCV20265 2464, SCV20265 _2518, SCV20265 _2654,
SCV20265 3101, SCV20265 _3909, SCV20265 _2610, SCV20265 _1805,
SCV20265 4445, SCV20265 _2883, SCV20265 2916, SCV20265 1721,
SCV20265 _3099, SCV20265 _1735, SCV20265 _6289, SCV20265 2974,
SCV20265 2404, SCV20265 _6135, SCV20265 _3626, SCV20265 _1050,
SCV20265 _0188, SCV20265 _5329, SCV20265 2792, SCV20265 _1617,
SCV20265 2236, SCV20265 0491, SCV20265 2422, SCV20265 _5463,
SCV20265 _5597, SCV20265 0241, SCV20265 4334, SCV20265 _0891,
SCV20265 _1756, SCV20265 1113, SCV20265 _1895, SCV20265 4827,
SCV20265 _4159, SCV20265 _4562, SCV20265 _3569, SCV20265 _0041,
SCV20265 0017, SCV20265 _0008, SCV20265 _0032, SCV20265 _0033,
SCV20265 _0007, SCV20265 _0028, SCV20265 0014, SCV20265 _0016.
According to certain embodiments, mutations in at least two, three, four, five, six, seven, eight, nine or ten genes are determined in any of the methods of the present invention, e.g. in at least two genes or in at least three genes. In¬ stead of testing only single genes or mutants, a combination of several variant positions can improve the prediction accu¬ racy and further reduce false positive findings that are in¬ fluenced by other factors. Therefore, it is in particular preferred to determine the presence of a mutation in 2, 3, 4, 5, 6, 7, 8 or 9 (or more) genes selected from Table 5.
Further, according to certain embodiments, the reference ge¬ nome of Pseudomonas is again NC_023149 as annotated at the NCBI . According to certain embodiments, statistical analysis in the present methods is carried out using Fisher' s test with p < 10~6, preferably p < 10~9, particularly p < 10~10. Al¬ so, according to certain embodiments, the method further comprises correlating different genetic sites to each other. Al- so the other aspects of the embodiments of the first and se¬ cond aspect of the invention apply.
Table 5: List of genes
Figure imgf000050_0001
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antimicrobial drug is an antibiotic. According to certain em- bodiments, the antibiotic is a lactam antibiotic and a muta¬ tion in at least one of the genes listed in Table 6 is de¬ tected, or a mutation in at least one of the positions (de¬ noted POS in the tables) listed in Table 6. Table 6: List for lactam antibiotics
gene name POS antibiotic p-value genbank protein
(FDR) accession num- ber
SCV20265_1892 1979239 T/Ξ ; CP; CFT ; LVX; GM; IMP ; ETP ; 1, 5814E-141 YP_008980900.1
MER; CAX; AZT; P/T; CPE; CAZ; TO
SCV20265_5625 5987559 T/Ξ ; CP; CFT ; LVX; GM; IMP ; ETP ; 1, 2153E-121 YP_008984625.1
MER; CAX; AZT; P/T; CPE; CAZ; TO
SCV20265_4334 4604211 T/Ξ ; CP; LVX; GM; ETP ; MER; AZT ; 2, 21898E-31 YP_008983338.1
P/T; CPE; TO
SCV20265_0891 938801 Τ/Ξ ; CP; GM; IMP; ETP; MER; P/T ; 2, 24131E-27 YP_008979899.1
TO ; LVX
SCV20265_1756 1846678 Τ/Ξ ; CP; GM; IMP; ETP; MER; P/T ; 2, 14003E-18 YP_008980764.1
TO ; LVX
SCV20265_1113 1169401 T/Ξ ; CP; GM; IMP ; ETP ; MER; TO; 2, 43872E-22 YP_008980121.1
LVX
SCV20265_1895 1984529 T/S;CP;LVX;GM;IMP;P/T;CPE; 1, 12601E-29 YP_008980903.1
TO
SCV20265_4827 5100757 T/S;CP;GM; IMP; MER; P/T ; TO; 2, 58406E-24 YP_008983827.1
LVX
SCV20265_1467 1537406 T/S;CP;LVX;GM;P/T;TO;CPE 1, 53793E-41 YP_008980475.1
SCV20265_2654 2754829 T/S;CP;LVX;GM;ETP;TO;CPE 1, 05377E-35 YP_008981662.1
SCV20265_6289 6702956 T/S;CP;LVX;GM;P/T;TO;CPE 1, 19545E-34 YP_008985285.1
SCV20265_3626 3881624 T/S;CP;LVX;GM;P/T;TO;CPE 2, 22675E-34 YP_008982632.1
SCV20265_1050 1099519 T/S;CP;LVX;GM;ETP;TO;CPE 2, 28786E-34 YP_008980058.1
SCV20265_4159 4419978 T/S;CP;LVX;GM;P/T;TO;CPE 1, 13138E-33 YP_008983163.1
SCV20265_4562 4832599 MER; ETP 2, 71389E-33 YP_008983564.1
FDR: determined according to FDR (Benjamini Hochberg) method (Benjamini
Hochberg, 1995)
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is IMP and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_0891 ,
SCV20265_1756, SCV20265_1113 , SCV20265_1895 , SCV20265_4827 is detected, or a mutation in at least one of the positions of
1979239, 5987559, 938801, 1846678, 1169401, 1984529, 5100757.
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is MER and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_4334,
SCV20265_0891, SCV20265_1756, SCV20265_1113 , SCV20265_4827 , SCV20265 4562 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 4604211, 938801, 1846678, 1169401, 5100757, 4832599.
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is ETP and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_4334,
SCV20265_0891, SCV20265_1756, SCV20265_1113 , SCV20265_1467 , SCV20265_2654, SCV20265_1050 , SCV20265_4562 is detected, or a mutation in at least one of the positions of 1979239,
5987559, 4604211, 938801, 1846678, 1169401, 1537406, 2754829, 1099519, 4832599. According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is P/T and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_4334,
SCV20265_0891, SCV20265_1756 , SCV20265_1895 , SCV20265_4827 ,
SCV20265_6289, SCV20265_3626, SCV20265_4159 is detected, or a mutation in at least one of the positions of 1979239,
5987559, 4604211, 938801, 1846678, 1984529, 5100757, 6702956, 3881624, 4419978.
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is CPE and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_4334,
SCV20265_1895, SCV20265_1467, SCV20265_2654 , SCV20265_6289 , SCV20265_3626, SCV20265_1050, SCV20265_4159 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 4604211, 1984529, 1537406, 2754829, 6702956,
3881624, 1099519, 4419978.
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is AZT and a mutation in at least one of the genes of SCV20265_1892, SCV20265_5625, SCV20265_4334 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 4604211.
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is at least one of CFT, CAX and CAZ and a mutation in at least one of the genes of SCV20265_1892 , SCV20265_5625 is detected, or a mutation in at least one of the positions ofl979239, 5987559. According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is a quinolone antibiotic and a mutation in at least one of the genes listed in Table 7 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 7.
Table 7: List for quinolone antibiotics
gene name POS antibiotic p-value genbank protein
(FDR) accession number
SCV20265_1892 1979239 T/S ; CP; CFT ; LVX; GM; IMP ; 1, 5814E-141 YP_008980900.1
ETP; MER; CAX; AZT; P/T; CPE;
CAZ; TO
SCV20265_5625 5987559 T/S ; CP; CFT ; LVX; GM; IMP ; 1, 2153E-121 YP_008984625.1
ETP; MER; CAX; AZT; P/T; CPE;
CAZ; TO SCV20265_1467 1537406 T/S;CP;LVX;GM;P/T;TO;CPE 1, 53793E-41 YP_008980475.1
SCV20265_5607 5965080 Τ/Ξ ; CP; GM; P/T ; LVX; TO 4, 52474E-39 YP_008984607.1
SCV20265_3294 3513162 T/S;LVX;CP;TO;GM 1, 34708E-37 YP_008982302.1
SCV20265_1879 1967346 Τ/Ξ ; CP; GM; P/T ; LVX; TO 1, 53005E-37 YP_008980887.1
SCV20265_5242 5569783 T/S;CP;LVX;GM;TO;CPE 2, 19789E-37 YP_008984242.1
SCV20265_2224 2350860 Τ/Ξ ; CP; GM; P/T ; LVX; TO 2, 41127E-37 YP_008981232.1
SCV20265_2224 2350862 Τ/Ξ ; CP; GM; P/T ; LVX; TO 2, 41127E-37 YP_008981232.1
SCV20265_0530 562872 T/S;CP;LVX;GM;TO;CPE 3, 4301E-37 YP_008979538.1
SCV20265_3289 3507580 T/S;CP;LVX;GM;TO;CPE 6, 35793E-37 YP_008982297.1
SCV20265_1858 1947689 T/S;CP;LVX;GM;TO;CPE 6, 77112E-37 YP_008980866.1
SCV20265_2193 2316386 T/S;CP;LVX;GM;TO;CPE 6, 77112E-37 YP_008981201.1
SCV20265_6274 6685845 T/S;CP;LVX;GM;TO;CPE 7, 04031E-37 YP_008985270.1
SCV20265_2958 3142437 T/S;CP;LVX;GM;TO;CPE 9, 40137E-37 YP_008981966.1
SCV20265_3248 3468647 T/S;CP;LVX;GM;TO;CPE 1, 00069E-36 YP_008982256.1
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is at least one of CP and LVX and a mutation in at least one of the genes of SCV20265_1892 , SCV20265_5625,
SCV20265_1467, SCV20265_5607 , SCV20265_3294 , SCV20265_1879 , SCV20265_5242, SCV20265_2224 , SCV20265_0530 , SCV20265_3289 , SCV20265_1858, SCV20265_2193 , SCV20265_6274 , SCV20265_2958 , SCV20265_3248 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 1537406, 5965080, 3513162, 1967346, 5569783, 2350860, 2350862, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647. According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is an aminoglycoside antibiotic and a mutation in at least one of the genes listed in Table 8 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 8.
Table 8: List of aminoglycoside antibiotics gene name POS antibiotic p-value genbank protein
(FDR) accession number
SCV20265_1892 1979239 T/S ; CP; CFT ; LVX; GM; IMP ; 1, 5814E-141 YP_008980900.1
ETP; MER; CAX; AZT; P/T; CPE;
CAZ; TO
SCV20265_5625 5987559 T/S ; CP; CFT ; LVX; GM; IMP ; 1, 2153E-121 YP_008984625.1
ETP; MER; CAX; AZT; P/T; CPE;
CAZ; TO
SCV20265_1467 1537406 T/S;CP;LVX;GM;P/T;TO;CPE 1, 53793E-41 YP_008980475.1
SCV20265_5607 5965080 T/S ; CP; GM; P/T ; LVX; TO 4, 52474E-39 YP_008984607.1
SCV20265_3294 3513162 T/S;LVX;CP;TO;GM 1, 34708E-37 YP_008982302.1
SCV20265_1879 1967346 T/S ; CP; GM; P/T ; LVX; TO 1, 53005E-37 YP_008980887.1
SCV20265_5242 5569783 T/S ; CP; LVX; GM; TO; CPE 2, 19789E-37 YP_008984242.1
SCV20265_2224 2350860 T/S ; CP; GM; P/T ; LVX; TO 2, 41127E-37 YP_008981232.1
SCV20265_2224 2350862 T/S ; CP; GM; P/T ; LVX; TO 2, 41127E-37 YP_008981232.1
SCV20265_0530 562872 T/S ; CP; LVX; GM; TO; CPE 3, 4301E-37 YP_008979538.1
SCV20265_3289 3507580 T/S ; CP; LVX; GM; TO; CPE 6, 35793E-37 YP_008982297.1
SCV20265_1858 1947689 T/S ; CP; LVX; GM; TO; CPE 6, 77112E-37 YP_008980866.1
SCV20265_2193 2316386 T/S ; CP; LVX; GM; TO; CPE 6, 77112E-37 YP_008981201.1
SCV20265_6274 6685845 T/S ; CP; LVX; GM; TO; CPE 7, 04031E-37 YP_008985270.1
SCV20265_2958 3142437 T/S ; CP; LVX; GM; TO; CPE 9, 40137E-37 YP_008981966.1
SCV20265_3248 3468647 T/S ; CP; LVX; GM; TO; CPE 1, 00069E-36 YP_008982256.1
According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is at least one of GM and TO and a mutation in at least one of the genes of SCV20265_1892 , SCV20265_5625,
SCV20265_1467, SCV20265_5607 , SCV20265_3294 , SCV20265_1879 , SCV20265_5242, SCV20265_2224 , SCV20265_0530 , SCV20265_3289 , SCV20265_1858, SCV20265_2193 , SCV20265_6274 , SCV20265_2958 , SCV20265_3248 is detected, or a mutation in at least one of the positions of 1979239, 5987559, 1537406, 5965080, 3513162, 1967346, 5569783, 2350860, 2350862, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647.
According to certain embodiments of the method of the seven¬ teenth and/or eighteenth aspect of the present invention, the antibiotic is T/S and a mutation in at least one of the genes listed in Table 9 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 12.
Table 9: List of others antibiotics ((benzene de- rived) /sulfonamide)
Figure imgf000056_0001
A fourteenth aspect of the present invention is directed to a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicro- bial drug treatment, which can also be described as method of determining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection of a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa- tient;
b) determining the presence of at least one mutation in at least one gene from the group of genes consisting of
SCV20265 1892, SCV20265 5625, SCV20265 1467, SCV20265 5607, SCV20265 3294, SCV20265 1879 , SCV20265 5242, SCV20265 2224,
SCV20265 _0530, SCV20265 3289 , SCV20265 _1858, SCV20265 2193,
SCV20265 6274, SCV20265 2958 , SCV20265 3248, SCV20265 1132,
SCV20265 1451, SCV20265 _6120 , SCV20265 _4839, SCV20265 2195,
SCV20265 _0968, SCV20265 2464 , SCV20265 _2518, SCV20265 2654,
SCV20265 3101, SCV20265 _3909 , SCV20265 _2610, SCV20265 1805,
SCV20265 4445, SCV20265 2883 , SCV20265 2916, SCV20265 1721,
SCV20265 _3099, SCV20265 _1735 , SCV20265 _6289, SCV20265 2974,
SCV20265 2404, SCV20265 _6135 , SCV20265 _3626, SCV20265 1050,
SCV20265 _0188, SCV20265 5329 , SCV20265 2792, SCV20265 1617,
SCV20265 2236, SCV20265 0491 , SCV20265 2422, SCV20265 5463,
SCV20265 _5597, and SCV20265 0241, wherein the presence of said at least one mutation is indicative of an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection in said patient.
A fifteenth aspect of the present invention is directed to a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least one gene from the group of genes consisting of
SCV20265_1892, SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_3294, SCV20265_1879 , SCV20265_5242 , SCV20265_2224 , SCV20265_0530, SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958 , SCV20265_3248 , SCV20265_1132 , SCV20265_1451, SCV20265_6120 , SCV20265_4839 , SCV20265_2195 , SCV20265_0968, SCV20265_2464, SCV20265_2518 , SCV20265_2654 , SCV20265_3101, SCV20265_3909 , SCV20265_2610 , SCV20265_1805 , SCV20265 4445, SCV20265 2883, SCV20265 2916, SCV20265 1721, SCV20265_3099, SCV20265_1735, SCV20265_6289, SCV20265_2974, SCV20265_2404, SCV20265_6135 , SCV20265_3626 , SCV20265_1050 , SCV20265_0188, SCV20265_5329, SCV20265_2792, SCV20265_1617 , SCV20265_2236, SCV20265_0491 , SCV20265_2422 , SCV20265_5463 , SCV20265_5597, and SCV20265_0241, wherein the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection.
Again, in the fourteenth and the fifteenth aspect the steps correspond to those in the first or second aspect, although only a mutation in at least one gene is determined.
A sixteenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, com¬ prising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least one gene from the group of genes consisting of
SCV20265_1892, SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_3294, SCV20265_1879 , SCV20265_5242 , SCV20265_2224 , SCV20265_0530, SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958 , SCV20265_3248 , SCV20265_1132 , SCV20265_1451, SCV20265_6120 , SCV20265_4839 , SCV20265_2195 , SCV20265_0968, SCV20265_2464, SCV20265_2518 , SCV20265_2654 , SCV20265 3101, SCV20265 3909, SCV20265 2610, SCV20265 1805, SCV20265_4445, SCV20265_2883 , SCV20265_2916 , SCV20265_1721 , SCV20265_3099, SCV20265_1735, SCV20265_6289, SCV20265_2974, SCV20265_2404, SCV20265_6135 , SCV20265_3626 , SCV20265_1050 , SCV20265_0188, SCV20265_5329, SCV20265_2792, SCV20265_1617 , SCV20265_2236, SCV20265_0491 , SCV20265_2422 , SCV20265_5463 , SCV20265_5597, and SCV20265_0241, wherein the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection; and e) treating the patient with said one or more antimicrobi- al, e.g. antibiotic, drugs.
A seventeenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobi¬ al drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least two genes from the group of genes consisting of
SCV20265_1892, SCV20265_5625 , SCV20265_1467 , SCV20265_5607 , SCV20265_3294, SCV20265_1879 , SCV20265_5242 , SCV20265_2224 , SCV20265_0530, SCV20265_3289, SCV20265_1858, SCV20265_2193 , SCV20265_6274, SCV20265_2958 , SCV20265_3248 , SCV20265_1132 , SCV20265_1451, SCV20265_6120 , SCV20265_4839 , SCV20265_2195 , SCV20265_0968, SCV20265_2464, SCV20265_2518 , SCV20265_2654 , SCV20265_3101, SCV20265_3909 , SCV20265_2610 , SCV20265_1805 , SCV20265 4445, SCV20265 2883, SCV20265 2916, SCV20265 1721, SCV20265_3099, SCV20265_1735, SCV20265_6289, SCV20265_2974, SCV20265_2404, SCV20265_6135 , SCV20265_3626 , SCV20265_1050 , SCV20265_0188, SCV20265_5329, SCV20265_2792, SCV20265_1617 , SCV20265_2236, SCV20265_0491 , SCV20265_2422 , SCV20265_5463 , SCV20265_5597, and SCV20265_0241, wherein the presence of said at least two mutations is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection; and e) treating the patient with said one or more antimicrobi¬ al, e.g. antibiotic, drugs.
An eighteenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobi¬ al drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 5, wherein the presence of said at least two mutations is indic¬ ative of a resistance to one or more antimicrobial, e.g. an¬ tibiotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection; and e) treating the patient with said one or more antimicrobi¬ al, e.g. antibiotic, drugs.
A nineteenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, com¬ prising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa- tient;
b) determining the presence of at least one mutation in at least one gene from the group of genes listed in Table 5, wherein the presence of said at least one mutation is indica¬ tive of a resistance to one or more antimicrobial, e.g. anti- biotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection; and e) treating the patient with said one or more antimicrobi¬ al, e.g. antibiotic, drugs.
Also in the sixteenth to nineteenth aspect of the invention, steps a) to d) are analogous to the steps in the method of the second aspect of the present invention. Step e) can be sufficiently carried out without being restricted and can be done e.g. non-invasively . A twentieth aspect of the present invention is directed to a diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicro¬ bial drug treatment, which can also be described as method of determining an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection of a patient, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa- tient;
b) determining the presence of at least one mutation in at least one gene from the group of genes listed in Table 5, wherein the presence of said at least one mutation is indica¬ tive of an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection in said patient.
A twenty-first aspect of the present invention is directed to a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Pseudomonas infection, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the pa¬ tient ;
b) determining the presence of at least one mutation in at least one gene from the group of genes listed in Table 5, wherein the presence of said at least one mutation is indica¬ tive of a resistance to one or more antimicrobial, e.g. anti¬ biotic, drugs;
c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection. Again, in the twentieth and the twenty-first aspect the steps correspond to those in the first or second aspect, although only a mutation in at least one gene is determined. Examples
The present invention will now be described in detail with reference to several examples thereof. However, these exam- pies are illustrative and do not limit the scope of the in¬ vention .
Example 1
Whole genome sequencing was carried out in addition to clas- sical antimicrobial susceptibility testing of the same iso¬ lates for a cohort of 1104 specimens. This allowed performing genome wide correlation studies to find genetic variants (e.g. point mutations, small insertions and deletion, larger structural variants, plasmid copy number gains, gene dosage effects) in the genome and plasmids that are significantly correlated to the resistance against one or several drugs. The approach also allows for comparing the relevant sites in the genome to each other. In the approach the different sources of genetic resistance as well as the different ways of how bacteria can become re¬ sistant were covered. By measuring clinical isolates collect¬ ed in a broad geographical area and across a broad time span of three decades a complete picture going far beyond the ra- ther artificial step of laboratory generated resistance mech¬ anisms was tried to be generated.
To this end, a set of 21 clinically relevant antimicrobial agents with 5 different modes of action was put together, and the minimally inhibitory concentration (MIC) of the 21 drugs for the Pseudomonas isolates was measured.
The detailed procedure is given in the following: Bacterial Strains
The inventors selected 1104 Pseudomonas strains, particularly Pseudomonas aeruginosa, from the microbiology strain collec¬ tion at Siemens Healthcare Diagnostics (West Sacramento, CA) for susceptibility testing and whole genome sequencing.
Antimicrobial Susceptibility Testing (AST) Panels
Frozen reference AST panels were prepared following Clinical
Laboratory Standards Institute (CLSI) recommendations. The following antimicrobial agents (with yg/ml concentrations shown in parentheses) were included in the panels: Amoxicil- lin/K Clavulanate (0.5/0.25-64/32), Ampicillin (0.25-128), Ampicillin/Sulbactam (0.5/0.25-64/32), Aztreonam (0.25-64), Cefazolin (0.5-32), Cefepime (0.25-64), Cefotaxime (0.25- 128), Ceftazidime (0.25-64), Ceftriaxone (0.25-128), Cefurox- ime (1-64), Cephalothin (1-64), Ciprofloxacin (0.015-8), Ertepenem (0.12-32), Gentamicin (0.12-32), Imipenem (0.25- 32), Levofloxacin (0.25-16), Meropenem (0.12-32),
Piperacillin/Tazobactam (0.25/4-256/4), Tetracycline (0.5- 64), Tobramycin (0.12-32), and Trimethoprim/Sulfamethoxazole (0.25/4.7-32/608). Prior to use with clinical isolates, AST panels were tested with QC strains. AST panels were consid¬ ered acceptable for testing with clinical isolates when the QC results met QC ranges described by CLSI16.
Inoculum Preparation
Isolates were cultured on trypticase soy agar with 5% sheep blood (BBL, Cockeysville, Md.) and incubated in ambient air at 35±1°C for 18-24 h. Isolated colonies (4-5 large colonies or 5-10 small colonies) were transferred to a 3 ml Sterile Inoculum Water (Siemens) and emulsified to a final turbidity of a 0.5 McFarland standard. 2 ml of this suspension was add- ed to 25 ml Inoculum Water with Pluronic-F (Siemens) . Using the Inoculator (Siemens) specific for frozen AST panels, 5 μΐ of the cell suspension was transferred to each well of the AST panel. The inoculated AST panels were incubated in ambi- ent air at 35±1°C for 16-20 h. Panel results were read visu¬ ally, and minimal inhibitory concentrations (MIC) were deter¬ mined .
DNA extraction
Four streaks of each Gram-negative bacterial isolate cultured on trypticase soy agar containing 5% sheep blood and cell suspensions were made in sterile 1.5 ml collection tubes con¬ taining 50 μΐ Nuclease-Free Water (AM9930, Life Technolo¬ gies) . Bacterial isolate samples were stored at -20 °C until nucleic acid extraction. The Tissue Preparation System (TPS) (096D0382-02_01_B, Siemens) and the VERSANT® Tissue Prepara¬ tion Reagents (TPR) kit (10632404B, Siemens) were used to ex¬ tract DNA from these bacterial isolates. Prior to extraction, the bacterial isolates were thawed at room temperature and were pelleted at 2000 G for 5 seconds. The DNA extraction protocol DNAext was used for complete total nucleic acid ex¬ traction of 48 isolate samples and eluates, 50 μΐ each, in 4 hours. The total nucleic acid eluates were then transferred into 96-Well qPCR Detection Plates (401341, Agilent Technolo- gies) for RNase A digestion, DNA quantitation, and plate DNA concentration standardization processes. RNase A (AM2271, Life Technologies) which was diluted in nuclease-free water following manufacturer's instructions was added to 50 μΐ of the total nucleic acid eluate for a final working concentra- tion of 20 μg/ml. Digestion enzyme and eluate mixture were incubated at 37 °C for 30 minutes using Siemens VERSANT® Am¬ plification and Detection instrument. DNA from the RNase digested eluate was quantitated using the Quant-iT™ PicoGreen dsDNA Assay (P11496, Life Technologies) following the assay kit instruction, and fluorescence was determined on the Sie¬ mens VERSANT® Amplification and Detection instrument. Data analysis was performed using Microsoft® Excel 2007. 25 μΐ of the quantitated DNA eluates were transferred into a new 96- Well PCR plate for plate DNA concentration standardization prior to library preparation. Elution buffer from the TPR kit was used to adjust DNA concentration. The standardized DNA eluate plate was then stored at -80°C until library prepara¬ tion .
Next Generation Sequencing
Prior to library preparation, quality control of isolated bacterial DNA was conducted using a Qubit 2.0 Fluorometer (Qubit dsDNA BR Assay Kit, Life Technologies) and an Agilent 2200 TapeStation (Genomic DNA ScreenTape, Agilent Technolo¬ gies) . NGS libraries were prepared in 96 well format using NexteraXT DNA Sample Preparation Kit and NexteraXT Index Kit for 96 Indexes (Illumina) according to the manufacturer's protocol. The resulting sequencing libraries were quantified in a qPCR-based approach using the KAPA SYBR FAST qPCR
MasterMix Kit (Peqlab) on a ViiA 7 real time PCR system (Life Technologies) . 96 samples were pooled per lane for paired-end sequencing (2x lOObp) on Illumina Hiseq2000 or Hiseq2500 se- quencers using TruSeq PE Cluster v3 and TruSeq SBS v3
sequncing chemistry (Illumina). Basic sequencing quality parameters were determined using the FastQC quality control tool for high throughput sequence data (Babraham Bioinformat- ics Institute) .
Data analysis
Raw paired-end sequencing data for the 1104 Pseudomonas sam¬ ples were mapped against the Pseudomonas reference (NC_023149) with BWA 0.6.1.20. The resulting SAM files were sorted, converted to BAM files, and PCR duplicates were marked using the Picard tools package 1.104
(https://picard.sourceforge.net/). The Genome Analysis Toolkit 3.1.1 (GATK)21 was used to call SNPs and indels for blocks of 200 Pseudomonas samples (parameters: -ploidy 1 -glm BOTH - stand_call_conf 30 -stand_emit_conf 10) . VCF files were combined into a single file and quality filtering for SNPs was carried out (QD < 2.0 | | FS > 60.0 | | MQ < 40.0) and indels (QD < 2.0 I I FS > 200.0). Detected variants were annotated with SnpEff22 to predict coding effects. For each annotated position, genotypes of all Pseudomonas samples were consid¬ ered. Pseudomonas samples were split into two groups, low re¬ sistance group (having lower MIC concentration for the con- sidered drug) , and high resistance group (having higher MIC concentrations) with respect to a certain MIC concentration (breakpoint) . To find the best breakpoint all thresholds were evaluated and p-values were computed with Fisher' s exact test relying on a 2x2 contingency table (number of Pseudomonas samples having the reference or variant genotype vs. number of samples belonging to the low and high resistance group) . The best computed breakpoint was the threshold yielding the lowest p-value for a certain genomic position and drug. For further analyses positions with non-synonymous alterations and p-value < 10 were considered.
Since a potential reason for drug resistance is gene duplica¬ tion, gene dose dependency was evaluated. For each sample the genomic coverage for each position was determined using BED Tools. Gene ranges were extracted from the reference assembly NC_023149. gff and the normalized median coverage per gene was calculated. To compare low- and high-resistance isolates the best area under the curve (AUC) value was computed. Groups of at least 20% of all samples having a median coverage larger than zero for that gene and containing more than 15 samples per group were considered in order to exclude artifacts and cases with AUC > 0.75 were further evaluated.
To include data on the different ways how resistance mecha¬ nisms are acquired Pseudomonas isolates collected over more than three decades were analyzed such that also horizontal gene transfer could potentially be discovered.
In detail, the following steps were carried out:
Pseudomonas strains to be tested were seeded on agar plates and incubated under growth conditions for 24 hours. Then, colonies were picked and incubated in growth medium in the presence of a given antibiotic drug in dilution series under growth conditions for 16-20 hours. Bacterial growth was de¬ termined by observing turbidity.
Next mutations were searched that are highly correlated with the results of the phenotypic resistance test.
For sequencing, samples were prepared using a Nextera library preparation, followed by multiplexed sequencing using the Illuminat HiSeq 2500 system, paired end sequencing. Data were mapped with BWA (Li H. and Durbin R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinfor- matics, Epub . [PMID: 20080505] ) and SNP were called using samtools (Li H.*, Handsaker B.*, Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Ge- nome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics , 25, 2078-9. [PMID: 19505943] ) . As reference genome, NC_0123149 as annotated at the NCBI was determined as best suited.
The mutations were matched to the genes and the amino acid changes were calculated. Using different algorithms (SVM, ho¬ mology modeling) mutations leading to amino acid changes with likely pathogenicity / resistance were calculated.
In total, whole genomes and plasmids of 1104 different clini- cal isolates of Pseudomonas species were sequenced, and clas¬ sical antimicrobial susceptibility testing (AST) against 21 therapy forms as described above was performed for all organ¬ isms. From the classical AST a table with 1104 rows (iso¬ lates) and 21 columns (MIC values for 21 drugs) was obtained. Each table entry contained the MIC for the respective isolate and the respective drug. The genetic data were mapped to dif¬ ferent reference genomes of Pseudomonas that have been anno¬ tated at the NCBI (https://www.ncbi.nlm.nih.gov/), and the best reference was chosen as template for the alignment - NC_023149 as annotated at the NCBI. Additionally, assemblies were carried out and it was verified that the sequenced ge¬ nomes fulfil all quality criteria to become reference ge¬ nomes . Next, genetic variants were evaluated. This approach resulted in a table with the genetic sites in columns and the same isolates in 1104 rows. Each table entry contained the genetic determinant at the respective site (A, C, T, G, small inser¬ tions and deletions, ...) for the respective isolate.
In a next step different statistical tests were carried out 1) For comparing resistance / susceptibility to genetic sites we calculated contingency tables and determined the significance using Fishers test
2) For comparing different sites to each other we calculat- ed the correlation between different genetic sites
3) For detecting gene dosage effects, e.g. loss or gain of genes (in the genome or on plasmids) we calculated the coverage (i.e. how many read map to the current posi¬ tion) at each site for resistant and not resistant iso- lates .
From the data, first the 50 genes with the best p-value were chosen for the list of mutations as well as the list of cor¬ related antibiotic resistance, representing Tables 1 and 2.
A full list of all genetic sites, drugs, drug classes, af¬ fected genes etc. is provided in Tables 3 and 4a, 4b and 4c, wherein Table 3 corresponds to Table 1 and represents the genes having the lowest p-values after determining mutations in the genes, and Table 4, respectively Tables 4a, 4b and 4c correspond to Table 2 and represent the genes having the low¬ est p-values after correlating the mutations with antibiotic resistance . In addition, the data with the best p-values for each antibi¬ otic class with the most antibiotic drugs, respectively, were evaluated, being disclosed in Tables 5 - 9.
In Tables 3 - 9 the columns are designated as follows:
Gene name: affected gene;
POS : genomic position of the SNP / variant in the Pseudomonas reference genome (see above) ; p-value: significance value calculated using Fishers exact test (determined according to FDR (Benjamini Hochberg) method (Benjamini Hochberg, 1995));
genbank protein accession number: (NCBI) Accession number of the corresponding protein of the genes
Also the antibiotic/drug classes, the number of significant antibiotics correlated to the mutations (over all antibiotics or over certain classes) , as well as the correlated antibiot- ics are denoted in the Tables.
201511268
71
Table 3: Detailed results for the genes in Example 1 (corresponding to Table
Figure imgf000072_0001
201511268
72
Figure imgf000073_0001
201511268
73
Figure imgf000074_0001
201511268
74
Figure imgf000075_0001
*: fluoroquinolone
Table 4a: Detailed results for the genes in Example 1 (corresponding to Table
Figure imgf000075_0002
201511268
75
Figure imgf000076_0001
1805165 T/S;CP;LVX;GM;TO;CPE 6 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone* ; Lactams
3299685 /S ; CP; GM; ETP; LVX; 0 6 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone* ; Lactams
1821163 T/S;CP;LVX;GM;TO;CPE 6 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone* ; Lactams
6702956 T/S;CP;LVX;GM; P/T;TO;CPE 7 other (benzene derived) /sulfonamide;
aminoglycoside; quinolone* ; Lactams
3160788 T/S;CP;LVX;GM;TO;CPE 6 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone* ; Lactams
2515627 T/S;LVX;CP;TO;GM 5 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone*
6535290 T/S;CP;LVX;GM;TO;CPE 6 other (benzene derived) /sulfonamide; 3 aminoglycoside; quinolone* ; Lactams
3881624 T/S;CP;LVX;GM; P/T;TO;CPE 7 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone* ; Lactams
1099519 T/S;CP;LVX;GM;ETP;TO;CPE 7 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone* ; Lactams
208902 T/S;LVX;CP;TO;GM 5 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone*
5662982 T/S;CP;LVX;GM;TO;CPE 6 other (benzene derived) /sulfonamide; 3 aminoglycoside; quinolone* ; Lactams
2903129 T/S;CP;LVX;GM;TO;CPE 6 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone* ; Lactams
1701758 T/S;LVX;CP;TO;GM 5 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone*
2363393 T/S;CP;LVX;GM;TO;CPE 6 other (benzene derived) /sulfonamide; 3 aminoglycoside; quinolone* ; Lactams
525701 T/S;LVX;CP;TO;GM 5 other (benzene derived) /sulfonamide; 4 aminoglycoside; quinolone*
2530203 T/S;LVX;CP;TO;GM 5 other (benzene derived) /sulfonamide; 3 aminoglycoside; quinolone*
5806684 T/S;LVX;CP;TO;GM 5 other (benzene derived) /sulfonamide; 3 aminoglycoside; quinolone*
5954547 T/S;LVX;CP;TO;GM 5 other (benzene derived) /sulfonamide; 3 aminoglycoside; quinolone*
201511268
77
Figure imgf000078_0001
*: fluoroquinolone
Table 4b: Detailed results for the genes in Example 1 (corresponding to Table 2, continued)
Figure imgf000078_0002
201511268
78
Figure imgf000079_0001
201511268
79
Figure imgf000080_0001
Table 4c: Detailed results for the genes in Example 1 (corresponding to Table 2, continued)
Figure imgf000080_0002
201511268
80
Figure imgf000081_0001
201511268
81
Figure imgf000082_0001
The p-value was calculated using the Fisher exact test based on contingency table with 4 fields: #samples Resistant / wild type; #samples Resistant / mutant; #samples not Resistant / wild type; #samples not Resistant / mutant
The test is based on the distribution of the samples in the 4 fields. Even distribution indicates no significance, while clustering into two fields indicates significance. The following results were obtained
- A total of 6,734 different correlations between genetic sites and anti-microbial agents were detected (p-value < 10~ 10).
- The biggest part of these were point mutations (i.e. single base exchanges)
- The highest significances (10~141 and 10~121) were reached for non-synonymous codings in YP_008980900.1 and
YP_008984625.1, respectively, particular in positions 1979239 and/or 5987559, respectively, with regard to reference genome NC_023149 as annotated at the NCBI; the mutation in position
1979239 with regard to reference genome NC_023149 as annotat¬ ed at the NCBI is a non-synonymous coding, particularly a co- don change aCc/aTc; aCc/aAc, and the mutation in position 5987559 with regard to reference genome NC_023149 as annotat- ed at the NCBI is a non-synonymous coding, particularly a co- don change tCg/tTg; tCg/tGg
- Besides these, insertions or deletions of up to four bases were discovered
- Further, potential genetic tests for four different drug classes relating to resistances were discovered
• β-lactams (includes Penicillins, Cephalosporins, Carbapenems, Monobactams ) • Quinolones, particularly Fluoroquinolones
• Aminoglycosides
• Folate synthesis inhibitors
- Potential genetic tests for the tested drugs/drug combina- tions were discovered:
Amoxicillin/Clavulanate, Ampicillin, Ampicillin/Sulbactam, Aztreonam, Cefazolin, Cefepime, Ceftazidime, Cefuroxime, Cephalothin, Imipenem, Piperacillin/Tazobactam, Ciprofloxacin, Levofloxacin, Gentamycin, Tobramycin, Tetracycline, Tri- methoprim/Sulfamethoxazol
- Mutations were observed in 2,757 different genes
A genetic test for the combined pathogen identification and antimicrobial susceptibility testing direct from the patient sample can reduce the time-to actionable result significantly from several days to hours, thereby enabling targeted treat¬ ment. Furthermore, this approach will not be restricted to central labs, but point of care devices can be developed that allow for respective tests. Such technology along with the present methods and computer program products could revolu¬ tionize the care, e.g. in intense care units or for admis¬ sions to hospitals in general. Furthermore, even applications like real time outbreak monitoring can be achieved using the present methods.
Instead of using only single variants, a combination of sev¬ eral variant positions can improve the prediction accuracy and further reduce false positive findings that are influ- enced by other factors.
Compared to approaches using MALDI-TOF MS, the present ap¬ proach has the advantage that it covers almost the complete genome and thus enables us to identify the potential genomic sites that might be related to resistance. While MALDI-TOF MS can also be used to identify point mutations in bacterial proteins, this technology only detects a subset of proteins and of these not all are equally well covered. In addition, the identification and differentiation of certain related strains is not always feasible.
The present method allows computing a best breakpoint for the separation of isolates into resistant and susceptible groups. The inventors designed a flexible software tool that allows to consider - besides the best breakpoints - also values de¬ fined by different guidelines (e.g. European and US guide¬ lines) , preparing for an application of the GAST in different countries.
The inventors demonstrate that the present approach is capa¬ ble of identifying mutations in genes that are already known as drug targets, as well as detecting potential new target sites.
The current approach enables
a. Identification and validation of markers for genetic
identification and susceptibility/resistance testing within one diagnostic test
b. validation of known drug targets and modes of action c. detection of potentially novel resistance mechanisms
leading to putative novel target / secondary target genes for new therapies

Claims

Claims
1. A diagnostic method of determining an infection of a patient with Pseudomonas species potentially resistant to antimicrobial drug, e.g. antibiotic, treatment, compris¬ ing the steps of:
a) obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the patient ;
b) determining the presence of at least one mutation in at least two genes from the group of genes consisting of
SCV20265 1892, SCV20265 _5625 , SCV20265 _1467,
SCV20265 _5607, SCV20265 3294 , SCV20265 _1879,
SCV20265 5242, SCV20265 2224 , SCV20265 _0530,
SCV20265 _3289, SCV20265 _1858 , SCV20265 _2193,
SCV20265 6274, SCV20265 _2958 , SCV20265 _3248,
SCV20265 1132, SCV20265 1451 , SCV20265 _6120,
SCV20265 _4839, SCV20265 _2195 , SCV20265 _0968,
SCV20265 2464, SCV20265 _2518 , SCV20265 _2654,
SCV20265 3101, SCV20265 _3909 , SCV20265 _2610,
SCV20265 _1805, SCV20265 4445 , SCV20265 _2883,
SCV20265 2916, SCV20265 1721 , SCV20265 _3099,
SCV20265 _1735, SCV20265 _6289 , SCV20265 2974,
SCV20265 2404, SCV20265 _6135 , SCV20265 _3626,
SCV20265 _1050, SCV20265 _0188 , SCV20265 _5329,
SCV20265 2792, SCV20265 1617 , SCV20265 _2236,
SCV20265 0491, SCV20265 2422 , SCV20265 _5463,
SCV20265 5597, and SCV20265 0241, wherein th
of said at least two mutations is indicative of an in¬ fection with an antimicrobial drug, e.g. antibiotic, re¬ sistant Pseudomonas strain in said patient.
2. A method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomo- nas strain, comprising the steps of:
obtaining or providing a sample containing or suspected of containing at least one Pseudomonas species from the patient ;
b) determining the presence of at least one mutation in at least two genes from the group of genes consisting of SCV20265 1892, SCV20265 5625, SCV20265 1467,
SCV20265_5607 SCV20265_3294 SCV20265 1879,
SCV20265_5242 SCV20265_2224 SCV20265 0530,
SCV20265_3289 SCV20265_1858 SCV20265 2193,
SCV20265_6274 SCV20265_2958 SCV20265 3248,
SCV20265_1132 SCV20265_1451 SCV20265 6120,
SCV20265_4839 SCV20265_2195 SCV20265 0968,
SCV20265_2464 SCV20265_2518 SCV20265 2654,
SCV20265_3101 SCV20265_3909 SCV20265 2610,
SCV20265_1805 SCV20265_4445 SCV20265 2883,
SCV20265_2916 SCV20265_1721 SCV20265 3099,
SCV20265_1735 SCV20265_6289 SCV20265 2974,
SCV20265_2404 SCV20265_6135 SCV20265 3626,
SCV20265_1050 SCV20265_0188 SCV20265 5329,
SCV20265_2792 SCV20265_1617 SCV20265 2236,
SCV20265_0491 SCV20265 2422 SCV20265 5463,
SCV20265 5597 and SCV20265_0241, wherein the presence of said at least two mutations is indicative of a re¬ sistance to one or more antimicrobial, e.g. antibiotic, drugs ;
identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and
d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infec¬ tion.
3. The method of one or more of the preceding claims,
wherein at least a mutation in SCV20265_1892 and/or SCV20265_5625, particularly in positions 1979239 and/or 5987559, respectively, with regard to reference genome NC_023149 as annotated at the NCBI, is determined.
4. The method of one or more of the preceding claims, where¬ in the method involves determining the resistance of Pseudomonas to one or more antimicrobial, e.g. antibi¬ otic, drugs.
5. The method of any one of claims 1 to 4, wherein the anti¬ microbial, e.g. antibiotic, drug is selected from lactam antibiotics and the presence of a mutation in the follow¬ ing genes is determined: SCV20265_1892 , SCV20265_5625,
SCV20265 1467, SCV20265 _5607, SCV20265 _1879,
SCV20265 5242, SCV20265 2224, SCV20265 _0530,
SCV20265 _3289, SCV20265 _1858, SCV20265 _2193,
SCV20265 6274, SCV20265 _2958, SCV20265 _3248,
SCV20265 1451, SCV20265 _6120, SCV20265 _4839,
SCV20265 _2195, SCV20265 _0968, SCV20265 2464,
SCV20265 _2518, SCV20265 _2654, SCV20265 _3101,
SCV20265 _1805, SCV20265 4445, SCV20265 _2883,
SCV20265 1721, SCV20265 _3099, SCV20265 _1735,
SCV20265 _6289, SCV20265 2974, SCV20265 _6135,
SCV20265 _3626, SCV20265 _1050, SCV20265 _5329,
SCV20265 2792, and/or SCV20265 2236; and/or
the antimicrobial, e.g. antibiotic, drug is selected from quinolone antibiotics, e.g. fluoroquinolone antibiotics, and the presence of a mutation in the following genes is determined: SCV20265 1892, SCV20265 5625, SCV20265 1467,
SCV20265 _5607, SCV20265 3294, SCV20265 _1879,
SCV20265 5242, SCV20265 2224, SCV20265 _0530,
SCV20265 _3289, SCV20265 _1858, SCV20265 _2193,
SCV20265 6274, SCV20265 _2958, SCV20265 _3248,
SCV20265 1132, SCV20265 1451, SCV20265 _6120,
SCV20265 _4839, SCV20265 _2195, SCV20265 _0968,
SCV20265 2464, SCV20265 _2518, SCV20265 _2654,
SCV20265 3101, SCV20265 _3909, SCV20265 _2610,
SCV20265 _1805, SCV20265 4445, SCV20265 _2883,
SCV20265 2916, SCV20265 1721, SCV20265 _3099,
SCV20265 _1735, SCV20265 _6289, SCV20265 2974,
SCV20265 2404, SCV20265 _6135, SCV20265 _3626,
SCV20265 _1050, SCV20265 _0188, SCV20265 _5329,
SCV20265 2792, SCV20265 1617, SCV20265 _2236,
SCV20265 0491, SCV20265 2422, SCV20265 _5463,
SCV20265 _5597, and/or SCV20265 0241; and/or
the antimicrobial, e.g. antibiotic, drug is selected from aminoglycoside antibiotics, and the presence of a muta¬ tion in the following genes is determined: SCV20265_1892 ,
SCV20265 _5625, SCV20265 1467, SCV20265 _5607,
SCV20265 3294, SCV20265 1879, SCV20265 5242,
SCV20265 2224, SCV20265 _0530, SCV20265 _3289,
SCV20265 _1858, SCV20265 2193, SCV20265 _6274,
SCV20265 _2958, SCV20265 3248, SCV20265 1132,
SCV20265 1451, SCV20265 _6120, SCV20265 _4839,
SCV20265 _2195, SCV20265 _0968, SCV20265 2464,
SCV20265 _2518, SCV20265 _2654, SCV20265 _3101,
SCV20265 _3909, SCV20265 _2610, SCV20265 _1805,
SCV20265 4445, SCV20265 _2883, SCV20265 _2916,
SCV20265 1721, SCV20265 _3099, SCV20265 _1735,
SCV20265 _6289, SCV20265 2974, SCV20265 2404,
SCV20265 6135, SCV20265 3626, SCV20265 1050, SCV20265_0188, SCV20265_5329, SCV20265_2792, SCV20265_1617, SCV20265_2236 , SCV20265_0491 ,
SCV20265_2422, SCV20265_5463 , SCV20265_5597 , and/or
SCV20265_0241; and/or
the antimicrobial, e.g. antibiotic, drug is selected from benzene derived/sulfonamide antibiotics, and the presence of a mutation in the following genes is determined:
SCV20265 1892, SCV20265 _5625, SCV20265 _1467,
SCV20265 _5607, SCV20265 3294, SCV20265 _1879,
SCV20265 5242, SCV20265 2224, SCV20265 _0530,
SCV20265 _3289, SCV20265 _1858, SCV20265 _2193,
SCV20265 6274, SCV20265 _2958, SCV20265 _3248,
SCV20265 1132, SCV20265 1451, SCV20265 _6120,
SCV20265 _4839, SCV20265 _2195, SCV20265 _0968,
SCV20265 2464, SCV20265 _2518, SCV20265 _2654,
SCV20265 3101, SCV20265 _3909, SCV20265 _2610,
SCV20265 _1805, SCV20265 4445, SCV20265 _2883,
SCV20265 2916, SCV20265 1721, SCV20265 _3099,
SCV20265 _1735, SCV20265 _6289, SCV20265 2974,
SCV20265 2404, SCV20265 _6135, SCV20265 _3626,
SCV20265 _1050, SCV20265 _0188, SCV20265 _5329,
SCV20265 2792, SCV20265 1617, SCV20265 _2236,
SCV20265 0491, SCV20265 2422, SCV20265 _5463,
SCV20265 _5597, and/or SCV20265 0241.
6. The method of one or more of the preceding claims, where¬ in the antimicrobial drug, e.g. antibiotic drug, is se¬ lected from the group consisting of Amoxicillin/K
Clavulanate (AUG) , Ampicillin (AM) , Aztreonam (AZT) , Cefazolin (CFZ) , Cefepime (CPE), Cefotaxime (CFT) ,
Ceftazidime (CAZ) , Ceftriaxone (CAX) , Cefuroxime (CRM), Cephalotin (CF) , Ciprofloxacin (CP) , Ertapenem (ETP) , Gentamicin (GM) , Imipenem (IMP), Levofloxacin (LVX) , Meropenem (MER) , Piperacillin/Tazobactam (P/T) , Ampicil- lin/Sulbactam (A/S) , Tetracycline (TE) , Tobramycin (TO), and Trimethoprim/Sulfamethoxazole (T/S).
The method of any one of claims 1 to 6, wherein the anti¬ biotic drug is T/S, CP, LVX, GM, and/or TO and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_023149:
1979239, 5987559, 1537406, 5965080, 3513162, 1967346, 5569783, 2350860, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647, 1194383, 1521674, 6520799, 5124971, 2317909, 1009933, 2567532, 2611669, 2754829, 3301233, 4166792, 2709322, 1899865, 4712288, 3019764, 3068671, 1805165, 3299685, 1821163, 6702956, 3160788, 2515627, 6535290, 3881624, 1099519, 208902, 5662982, 2903129, 1701758, 2363393, 525701, 2530203, 5806684, 5954547, 261978, 2350862, 3507601, 3507667, 6519971;
and/or
wherein the antibiotic drug is CPE and a mutation in at least one of the following nucleotide positions is de¬ tected with regard to reference genome NC_023149:
1979239, 5987559, 1537406, 5569783, 562872, 3507580, 1947689, 2316386, 6685845, 3142437, 3468647, 1521674, 6520799, 5124971, 2317909, 1009933, 2567532, 2611669, 2754829, 1899865, 4712288, 1805165, 1821163, 6702956, 3160788, 6535290, 3881624, 1099519, 5662982, 2903129, 2363393, 3507601, 3507667, 6519971; and/or
wherein the antibiotic drug is P/T and a mutation in at least one of the following nucleotide positions is de¬ tected with regard to reference genome NC_023149:
1979239, 5987559, 1537406, 5965080, 1967346, 2350860, 2754829, 3301233, 3019764, 6702956, 3881624; and/or wherein the antibiotic drug is ETP and a mutation in at least one of the following nucleotide positions is de¬ tected with regard to reference genome NC_023149:
1979239, 5987559, 2754829, 3301233, 3299685, 1099519, 2350862; and/or
wherein the antibiotic drug is CFT, IMP, MER, CAX, AZT, and/or CAZ and a mutation in at least one of the following nucleotide positions is detected with regard to ref¬ erence genome NC_023149: 1979239, 5987559.
The method of any one of claims 1 to 7, wherein the re¬ sistance of a bacterial microorganism belonging to the species Pseudomonas against 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, 17, 18, 19, 20 or 21 anti biotic drugs is determined.
The method of one or more of the preceding claims, where in determining the nucleic acid sequence information or the presence of a mutation comprises determining a par¬ tial sequence or an entire sequence of the at least two genes .
The method of one or more of the preceding claims, where¬ in determining the nucleic acid sequence information or the presence of a mutation comprises determining a par¬ tial or entire sequence of the genome of the Pseudomonas species, wherein said partial or entire sequence of the genome comprises at least a partial sequence of said at least two genes.
The method of one or more of the preceding claims, where¬ in determining the nucleic acid sequence information or the presence of a mutation comprises using a next genera- tion sequencing or high throughput sequencing method, preferably wherein a partial or entire genome sequence the bacterial organism of Pseudomonas species is deter mined by using a next generation sequencing or high throughput sequencing method.
A method of determining an antimicrobial drug, e.g. anti¬ biotic, resistance profile for bacterial microorganisms of Pseudomonas species, comprising:
obtaining or providing a first data set of gene sequences of a plurality of clinical isolates of Pseudomonas spe¬ cies;
providing a second data set of antimicrobial drug, e.g. antibiotic, resistance of the plurality of clinical iso¬ lates of Pseudomonas species;
aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Pseudomo¬ nas, and/or assembling the gene sequence of the first da¬ ta set, at least in part;
analyzing the gene sequences of the first data set for genetic variants to obtain a third data set of genetic variants ;
correlating the third data set with the second data set and statistically analyzing the correlation; and
determining the genetic sites in the genome of Pseudomo¬ nas associated with antimicrobial drug, e.g. antibiotic, resistance .
13. A diagnostic method of determining an infection of a pa¬ tient with Pseudomonas species potentially resistant to antimicrobial drug treatment, comprising the steps of: a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Pseudomonas from the patient;
b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism be¬ longing to the species Pseudomonas as determined by the method of claim 12, wherein the presence of said at least one mutation is indicative of an infection with an antimicrobial drug resistant Pseudomonas strain in said pa- tient.
A method of selecting a treatment of a patient suffering from an infection with a potentially resistant Pseudomo¬ nas strain, comprising the steps of:
a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Pseudomonas from the patient;
b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism be¬ longing to the species Pseudomonas as determined by the method of claim 12, wherein the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial drugs;
c) identifying said at least one or more antimicrobial drugs; and
d) selecting one or more antimicrobial drugs different from the ones identified in step c) and being suitable for the treatment of a Pseudomonas infection. 15. A method of acquiring an antimicrobial drug, e.g. antibi¬ otic, resistance profile for bacterial microorganisms of Pseudomonas species, comprising: obtaining or providing a first data set of gene sequences of a clinical isolate of Pseudomonas species;
providing a second data set of antimicrobial drug, e.g. antibiotic, resistance of a plurality of clinical iso¬ lates of Pseudomonas species;
aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Pseudomo¬ nas, and/or assembling the gene sequence of the first da¬ ta set, at least in part;
analyzing the gene sequences of the first data set for genetic variants to obtain a third data set of genetic variants of the first data set;
correlating the third data set with the second data set and statistically analyzing the correlation; and
determining the genetic sites in the genome of Pseudomo¬ nas of the first data set associated with antimicrobial drug, e.g. antibiotic, resistance.
16. Computer program product comprising computer executable instructions which, when executed, perform a method ac¬ cording to any one of claims 12 to 15.
PCT/EP2015/066773 2015-07-22 2015-07-22 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents WO2017012661A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
PCT/EP2015/066773 WO2017012661A1 (en) 2015-07-22 2015-07-22 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents
PCT/EP2016/067406 WO2017013204A1 (en) 2015-07-22 2016-07-21 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents
CN201680038539.3A CN108513589A (en) 2015-07-22 2016-07-21 Genetic test for the resistance for predicting pseudomonad species combating microorganisms agent
CA2990908A CA2990908A1 (en) 2015-07-22 2016-07-21 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents
AU2016295122A AU2016295122A1 (en) 2015-07-22 2016-07-21 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents
US15/745,633 US20180265913A1 (en) 2015-07-22 2016-07-21 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents
EP16745655.7A EP3325655A1 (en) 2015-07-22 2016-07-21 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2015/066773 WO2017012661A1 (en) 2015-07-22 2015-07-22 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents

Publications (1)

Publication Number Publication Date
WO2017012661A1 true WO2017012661A1 (en) 2017-01-26

Family

ID=53762163

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/EP2015/066773 WO2017012661A1 (en) 2015-07-22 2015-07-22 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents
PCT/EP2016/067406 WO2017013204A1 (en) 2015-07-22 2016-07-21 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/067406 WO2017013204A1 (en) 2015-07-22 2016-07-21 Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents

Country Status (6)

Country Link
US (1) US20180265913A1 (en)
EP (1) EP3325655A1 (en)
CN (1) CN108513589A (en)
AU (1) AU2016295122A1 (en)
CA (1) CA2990908A1 (en)
WO (2) WO2017012661A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180216167A1 (en) * 2015-07-29 2018-08-02 Ares Genetics Gmbh Genetic testing for predicting resistance of stenotrophomonas species against antimicrobial agents

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010511408A (en) * 2006-12-04 2010-04-15 ザ・ボード・オブ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・イリノイ Compositions and methods for treating cancer with CpG rich DNA and cupredoxins
US11572561B2 (en) * 2020-06-19 2023-02-07 City University Of Hong Kong Method and composition for inhibiting growth of bacterium
CN113744806B (en) * 2021-06-23 2024-03-12 杭州圣庭医疗科技有限公司 Fungus sequencing data identification method based on nanopore sequencer
CN118006813A (en) * 2023-09-14 2024-05-10 北京金匙医学检验实验室有限公司 Characteristic gene for predicting drug sensitivity phenotype of pseudomonas aeruginosa carbapenem drug

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006097232A2 (en) * 2005-03-18 2006-09-21 Eppendorf Ag Antibiotic susceptibility and virulence factor detection in pseudomonas aeruginosa
WO2012027302A2 (en) * 2010-08-21 2012-03-01 The Regents Of The University Of California Systems and methods for detecting antibiotic resistance
US20120071336A1 (en) * 2010-06-02 2012-03-22 Medical Diagnostic Laboratories, Llc Antibiotic resistance profile for Neisseria gonorrhoeae and use of same in diagnosis and treatment of gonorrhea
WO2012106432A2 (en) * 2011-02-01 2012-08-09 Baylor College Of Medicine A genomic approach to the identification of biomarkers for antibiotic resistance and susceptibility in clinical isolates of bacterial pathogens

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006097232A2 (en) * 2005-03-18 2006-09-21 Eppendorf Ag Antibiotic susceptibility and virulence factor detection in pseudomonas aeruginosa
US20120071336A1 (en) * 2010-06-02 2012-03-22 Medical Diagnostic Laboratories, Llc Antibiotic resistance profile for Neisseria gonorrhoeae and use of same in diagnosis and treatment of gonorrhea
WO2012027302A2 (en) * 2010-08-21 2012-03-01 The Regents Of The University Of California Systems and methods for detecting antibiotic resistance
WO2012106432A2 (en) * 2011-02-01 2012-08-09 Baylor College Of Medicine A genomic approach to the identification of biomarkers for antibiotic resistance and susceptibility in clinical isolates of bacterial pathogens

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CLAIRE CHEWAPREECHA ET AL: "Comprehensive Identification of Single Nucleotide Polymorphisms Associated with Beta-lactam Resistance within Pneumococcal Mosaic Genes", PLOS GENETICS, vol. 10, no. 8, 7 August 2014 (2014-08-07), pages e1004547, XP055204428, DOI: 10.1371/journal.pgen.1004547 *
MICHAL WOZNIAK ET AL: "An approach to identifying drug resistance associated mutations in bacterial strains", BMC GENOMICS, BIOMED CENTRAL LTD, LONDON, UK, vol. 13, no. Suppl 7, 7 December 2012 (2012-12-07), pages S23, XP021127984, ISSN: 1471-2164, DOI: 10.1186/1471-2164-13-S7-S23 *
N. STOESSER ET AL: "Predicting antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data", JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, vol. 4, 30 May 2013 (2013-05-30), XP055157002, ISSN: 0305-7453, DOI: 10.1093/jac/dkt180 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180216167A1 (en) * 2015-07-29 2018-08-02 Ares Genetics Gmbh Genetic testing for predicting resistance of stenotrophomonas species against antimicrobial agents

Also Published As

Publication number Publication date
US20180265913A1 (en) 2018-09-20
CA2990908A1 (en) 2017-01-26
EP3325655A1 (en) 2018-05-30
WO2017013204A1 (en) 2017-01-26
AU2016295122A1 (en) 2018-01-25
CN108513589A (en) 2018-09-07

Similar Documents

Publication Publication Date Title
US20190085377A1 (en) Genetic testing for predicting resistance of salmonella species against antimicrobial agents
US20190093148A1 (en) Genetic testing for predicting resistance of serratia species against antimicrobial agents
EP3325659A2 (en) Genetic testing for predicting resistance of gram-negative proteus against antimicrobial agents
US20180265913A1 (en) Genetic testing for predicting resistance of pseudomonas species against antimicrobial agents
WO2016045857A1 (en) Genetic testing for predicting resistance of klebsiella species against antimicrobial agents
US20180363030A1 (en) Genetic testing for predicting resistance of enterobacter species against antimicrobial agents
US20180201979A1 (en) Genetic testing for predicting resistance of acinetobacter species against antimicrobial agents
US20180216167A1 (en) Genetic testing for predicting resistance of stenotrophomonas species against antimicrobial agents
US20180223336A1 (en) Genetic testing for predicting resistance of morganella species against antimicrobial agents
EP3101140A1 (en) Genetic testing for predicting resistance of shigella species against antimicrobial agents

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15744528

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15744528

Country of ref document: EP

Kind code of ref document: A1